Detection of differences in nucleic acids

ABSTRACT

A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both strands of each sequence. Each member of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of a difference between the two related sequences. The complex generally comprises a Holliday junction. In one aspect a medium suspected of containing said two related nucleic acid sequences is treated to provide partial duplexes having non-complementary tailed portions at one end. The double stranded portions of the partial duplexes are identical except for said difference. One of the strands of one of the partial duplexes is complementary to one of the strands of the other of the partial duplexes and the other of the strands of one of the partial duplexes is complementary to the other of the strands of the other of the partial duplexes. The medium is subjected to conditions that permit the binding of the tailed portions of the partial duplexes to each other. If there is a difference in the related nucleic acid sequences, a stable complex is formed comprising a Holliday junction. If no difference exists, the complex dissociates into duplexes. A determination is made whether the stable complex is formed, the presence thereof indicating the presence of the related nucleic acid sequences. The method has application in detecting the presence of a mutation in a target sequence or in detecting the target sequence itself.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] Nucleic acid hybridization has been employed for investigatingthe identity and establishing the presence of nucleic acids.Hybridization is based on complementary base pairing. When complementarysingle stranded nucleic acids are incubated together, the complementarybase sequences pair to form double stranded hybrid molecules. Theability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleicacid (RNA) to form a hydrogen bonded structure with a complementarynucleic acid sequence has been employed as an analytical tool inmolecular biology research. The availability of radioactive nucleosidetriphosphates of high specific activity and the ³²p labeling of DNA withT4 polynucleotide kinase has made it possible to identify, isolate, andcharacterize various nucleic acid sequences of biological interest.

[0003] Nucleic acid hybridization has great potential in diagnosingdisease states associated with unique nucleic acid sequences. Theseunique nucleic acid sequences may result from genetic or environmentalchange in DNA by insertions, deletions, point mutations, or by acquiringforeign DNA or RNA by means of infection by bacteria, molds, fungi, andviruses. Nucleic acid hybridization has, until now, been employedprimarily in academic and industrial molecular biology laboratories. Theapplication of nucleic acid hybridization as a diagnostic tool inclinical medicine is limited because of the frequently very lowconcentrations of disease related DNA or RNA present in a patient's bodyfluid and the unavailability of a sufficiently sensitive method ofnucleic acid hybridization analysis.

[0004] One method for detecting specific nucleic acid sequencesgenerally involves immobilization of the target nucleic acid on a solidsupport such as nitrocellulose paper, cellulose paper, diazotized paper,or a nylon membrane. After the target nucleic acid is fixed on thesupport, the support is contacted with a suitably labeled probe nucleicacid for about two to forty-eight hours. After the above time period,the solid support is washed several times at a controlled temperature toremove unhybridized probe. The support is then dried and the hybridizedmaterial is detected by autoradiography or by spectrometric methods.

[0005] When very low concentrations must be detected, the above methodis slow and labor intensive, and nonisotopic labels that are lessreadily detected than radiolabels are frequently not suitable.

[0006] A method for the enzymatic amplification of specific segments ofDNA known as the polymerase chain reaction (PCR) method has beendescribed. This in vitro amplification procedure is based on repeatedcycles of denaturation, oligonucleotide primer annealing, and primerextension by thermophilic polymerase, resulting in the exponentialincrease in copies of the region flanked by the primers. The PCRprimers, which anneal to opposite strands of the DNA, are positioned sothat the polymerase catalyzed extension product of one primer can serveas a template strand for the other, leading to the accumulation of adiscrete fragment whose length is defined by the distance between thehybridization sites on the DNA sequence complementary to the 5′ ends ofthe oligonucleotide primers.

[0007] Other methods for amplifying nucleic acids are single primeramplification, ligase chain reaction (LCR), nucleic acid sequence basedamplification (NASBA) and the Q-beta-replicase method. Regardless of theamplification used, the amplified product must be detected.

[0008] Genetic recombination involves the exchange of DNA strandsbetween two related DNA duplexes. The branch point between two duplexDNAs that have exchanged a pair of strands is thought to be an importantintermediate in homologous recombination. This branch point is otherwisereferred to as the Holliday junction. Movement of the Holliday junctionby branch migration can increase or decrease the amount of geneticinformation exchanged between homologues. In vitro strand exchange isprotein mediated, unlike the spontaneous migration that occurs in vitro.

[0009] There is a great demand for simple universal high-throughputmethods for detection of differences in related nucleic acid sequencesregardless of the exact nature of the difference. This demand isbecoming more and more urgent due to the ongoing rapid discovery of newdisease related mutations brought about by the progress of the HumanGenome Project. A detection method for mutations that is not dependenton the exact location of the mutation is valuable in the case ofdiseases that are known to result from various mutations within a givensequence. Moreover, such a method will be useful for verification ofsequence homology as related to various applications in molecularbiology, molecular medicine and population genetics.

[0010] Some of the current methods are either targeted for sets of knownmutations, such as, for example, the Reverse Dot Blot method, or involvegel-based techniques, such as, for example, single strandedconformational polymorphism (SSCP), denaturing gradient gelelectrophoresis (DGGE) or direct sequencing as well as a number ofmethods for the detection of heteroduplexes. Accordingly, such methodsare laborious and time consuming.

[0011] Various methods for mutation detection have been developed in therecent years based on amplification technology. The detection ofsequence alterations is based on one of the following principles:allele-specific hybridization, chemical modification of mismatched baseswith subsequent strand cleavage, nuclease cleavage at mismatches,recognition of mismatches by specific DNA binding proteins, changes inelectrophoretic mobility of mismatched duplexes in gradients ofdenaturing agents, conformation-induced changes in electrophoreticmobility of single-stranded DNA sometimes combined withconformation-specific nuclease cleavage. Some of these methods are toolaborious and time-consuming and many depend on the nature of basealteration.

[0012] It is desirable to have a sensitive, simple, inexpensive methodfor detecting differences in nucleic acids such as mutations,preferably, in a homogeneous format. The method should minimize thenumber and complexity of steps and reagents. Such a method would besuitable for a large scale population screening.

[0013] 2. Description of the Related Art

[0014] Formation of a single base mismatch that impedes spontaneous DNAbranch migration is described by Panyutin, et al., (1993) J. Mol. Biol.,230:413-424.

[0015] The kinetics of spontaneous DNA branch migration is discussed byPanyutin, et al., (1994) Proc. Natl. Acad. Sci. USA, 91: 2021-2025.

[0016] European Patent Application No. 0 450 370 Al (Wetmur, et al.,)discloses branch migration of nucleotides.

[0017] A displacement polynucleotide assay method and polynucleotidecomplex reagent therefor is discussed in U.S. Pat. No. 4, 766,062(Diamond, et al.,).

[0018] A strand displacement assay and complex useful therefor isdiscussed in PCT application WO 94/06937 (Eadie, et al.,).

[0019] PCT application WO/86/06412 (Fritsch, et al.,) discusses processand nucleic acid construct for producing reagent complexes useful indetermining target nucleotide sequences.

[0020] A process for amplifying, detecting and/or cloning nucleic acidsequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159, 4,965,188 and 5,008,182. Sequence polymerization bypolymerase chain reaction is described by Saiki, et al., (1986) Science,230: 1350-1354. Primer-directed enzymatic amplification of DNA with athermostable DNA polymerase is described by Saiki, et al, Science (1988)239:487.

SUMMARY OF THE INVENTION

[0021] One method in accordance with the present invention is directedto the detection of the presence of a difference between two relatednucleic acid sequences. In the method a complex is formed comprisingboth of the nucleic acid sequences in double stranded form. Both membersof at least one pair of non-complementary strands within the complexhave labels. The association of the labels as part of the complex isdetermined as an indication of the presence of the difference betweenthe two related sequences. The complex may comprise a Holliday junction.The complex does not dissociate at least until detection of the labelsas part of the complex has taken place and therefore, is in that sense astable complex.

[0022] Another embodiment of the present invention is a method fordetecting the presence of a difference between two related nucleic acidsequences. A medium suspected of containing two related nucleic acidsequences is treated to provide two partial duplexes each comprised offully matched duplexes having at one end non-complementary end portions.The partial duplexes are related in that, except for the difference, oneof the strands S1 of one of the partial duplexes is complementary to oneof the strands S1′ of the other of the partial duplexes and the other ofthe strands S2 of one of the partial duplexes is complementary to theother of the strands S2′ of the other of the partial duplexes. Themedium is subjected to conditions that permit the binding of S1 to S1′and S2 to S2′, respectively. If the medium contains a difference betweenthe related nucleic acid sequences, a stable complex is formedcomprising strands S1, S1′, S2 and S2′. A determination is made whetherthe stable complex is formed, the presence thereof indicating thepresence of a difference between the related nucleic acid sequences.

[0023] Another aspect of the present invention is a method for detectinga mutation within a target nucleic acid sequence. The method comprisesforming from the target sequence a tailed target partial duplexcomprising a duplex of the target sequence, a label and at one end ofthe duplex, two non-complementary oligonucleotides, one linked to eachstrand. The tailed target partial duplex is provided in combination witha labeled tailed reference partial duplex lacking the mutation. Thetailed reference partial duplex is comprised of two nucleic acid strandsthat are complementary to the strands in the tailed target partialduplex but for the possible presence of a mutation. Labels are presentin non-complementary strands of the tailed target and tailed referencepartial duplexes, respectively. The formation of a stable complexbetween the tailed partial duplexes is detected by means of the labels.The formation of the complex is directly related to the presence of themutation.

[0024] Another aspect of the present invention is a method of detectinga mutation within a target nucleic acid sequence, which is firstamplified by the polymerase chain reaction using primers P1 and P2 toproduce an amplicon AA. At least one of the primers P1 and P2 contains alabel and primer P1 is comprised of a 3′-end portion Pa that canhybridize with the target sequence and 5′-end portion B1 that cannothybridize with the target sequence. A chain extension of a primer P3along one strand of amplicon AA is carried out to produce a tailedtarget partial duplex A′. Primer P3 is comprised of 3′-end portion Paand a 5′-end portion A1 that cannot hybridize to the target sequence. Areference nucleic acid sequence is also amplified, using primer P2 andprimer P3, by polymerase chain reaction to produce amplicon BB. Thereference sequence is identical to the target sequence but lacking apossible mutation. Generally, when primer P2 used in the amplificationof the target nucleic acid sequence comprises a label, primer P2 usedfor amplification of the reference nucleic acid sequence comprises alabel that may be the same as or different than the label of primer P2.When primer P1 used for amplification of the target nucleic acidsequence comprises a label, primer P3 comprises a label that may be thesame as or different than the label of primer P1. A chain extension ofprimer P1 along one strand of amplicon BB is carried out to produce atailed reference partial duplex B′, which is allowed to bind to thetailed target partial duplex A′. The binding of one labeled strand tothe other labeled strand as a result of the formation of a stablecomplex between the tailed partial duplexes is detected, the bindingthereof being directly related to the presence of the mutation.

[0025] Another aspect of the present invention is a method for detectinga mutation in a nucleic acid, wherein a partial duplex A′ is producedfrom a target nucleic acid sequence suspected of having a mutation.Partial duplex A′ comprises a fully complementary double strandednucleic acid sequence containing the target nucleic acid sequence. Onestrand thereof has at its 5″-end a portion A1 that does not hybridizewith a corresponding portion A2 at the 3′-end of the other strand. Atleast one of the strands of the partial duplex A′ comprises a label. Apartial duplex B′ is produced from a reference nucleic acid sequencethat corresponds to the target nucleic acid sequence except for themutation. Partial duplex B′ comprises the double stranded nucleic acidsequence lacking the mutation wherein the strand corresponding to thestrand comprising the portion A1 has at its 5′-end a portion B1 that iscomplementary with A2 and the other strand has at its 3′-end a portionB2 that is complementary with A1. One of the strands of partial duplexB′ comprises a label wherein such strand is unable to hybridize directlyto the strand of partial duplex A′ that comprises a label. Partialduplexes A′ and B′ are subjected to conditions that permit the duplexesto hybridize to each other. If the target nucleic acid sequence havingthe mutation is present, a stable complex is formed comprising partialduplex A′ and partial duplex B′, the presence thereof indicating thepresence of the nucleic acid having the mutation.

[0026] Another embodiment of the present invention is a method fordetecting a mutation in a target nucleic acid. A medium containing (i)the target nucleic acid suspected of having the mutation and (ii) twoprimers P1 and P2, wherein P1 is extendable along one of the strands ofthe nucleic acid, is subjected to temperature cycling in the presence ofa nucleotide polymerase and nucleoside triphosphates. P1 has a 3′-endportion Pa that does bind, and a 5′-end portion B1 that does not bind,to one of the target nucleic acid strands. P2 is extendable along theother of the strands of the target nucleic acid. In this way oneextended primer is a template for the other of the primers. The mediumis then combined with a primer P3, which has the 3′-end portion Pa and a5′-end portion Al that does not bind to the extended P2 primer. Themedium is then subjected to conditions such that P3 is extended alongextended primer P2 to produce only a complementary strand, and not acopy, thereof. A medium containing (i) a reference nucleic acid, whichhas an identical sequence to the sequence of the target nucleic acidexcept for the mutation and (ii) two primers P3 and P2 is subjected totemperature cycling in the presence of a nucleotide polymerase andnucleoside triphosphates. P3 is extendable along one of the strands ofthe reference nucleic acid, wherein Pa binds, and Al does not bind,thereto. P2 is extendable along the other of the strands of thereference nucleic acid. Extended primer produced by the extension of oneof the primers is a template for the other of the primers. The lattermedium is combined with primer P1 wherein Pa binds to, and B1 does notbind to, the extended primer produced by extending P2 along thereference nucleic acid. The latter medium is subjected to conditionsunder which P1 is extended along extended primer P2 to produce only acomplementary strand, and not a copy, of the extended primer P2. Theabove steps may be carried out together in the same medium or inseparate reaction media and may be carried out simultaneously or whollyor partially sequentially. If the reactions are carried out in separatereaction media, the media are combined and subjected to conditions thatpermit the complementary strands produced in the above steps to bind tothe extended primers P1 and P3, respectively, such that a stable complexis formed if a mutation is present in the target nucleic acid. Adetermination is made as to whether such complex is formed, the presencethereof indicating the presence of the mutation.

[0027] Another aspect of the present invention concerns a method ofpreparing a DNA partial duplex having a portion at an end thereof thathas two predefined non-complementary single stranded sequences. A mediumcontaining a nucleic acid is combined with a polymerase, nucleosidetriphosphates and two primers. One of the primers P3 is extendable alongone of the strands of the nucleic acid. P3 has a 3′-end portion Pa thatdoes bind, and a 5′-end portion A1 that does not bind, thereto. Theother of the primers P2 is extendable along the other of the strands ofthe nucleic acid. Extended primer produced by the extension of one ofthe primers is a template for the other of the primers. The medium issubjected to temperature cycling to extend the primers and then combinedwith a primer P1, which has 3′-end portion Pa that binds, and a 5′-endportion B1 that does not bind, to the extended primers. The medium issubjected to conditions such that P3 binds to and is extended along theextended primer P2 to produce only a complement, and not a copy, of theextended primer.

[0028] Another aspect of the present invention is a method of preparinga DNA partial homoduplex having a portion at one end that has twonon-complementary single stranded sequences. A medium containing asingle stranded polynucleotide is combined with a primer P1 wherein P1has a 3′-end portion Pa that binds to a sequence that is 8 to 60nucleotides from the 3′-end of the single stranded polynucleotide and a8 to 60 nucleotide portion B1 that does not bind to the single strandedpolynucleotide. The medium is subjected to conditions under which P1binds to and is extended along the single stranded polynucleotide.

[0029] Another aspect of the present invention is a method for detectinga difference between two related nucleic acid sequences. A stablequadramolecular complex is formed comprising both of the nucleic acidsequences in double stranded form. The presence of the stable complex isdetected by binding the complex to a receptor. The presence of thestable complex indicates the presence of a difference between the tworelated sequences.

[0030] The present invention also includes kits for determining a targetnucleic acid sequence. A kit in accordance with the present inventioncomprises in packaged combination (a) a primer P2 that is extendablealong one of the strands of the target nucleic acid, (b) a primer P1comprising a 3′-end portion Pa that binds to and is extendable along theother of the strands of the target nucleic acid and a 5′-end portion B1that does not bind to the target nucleic acid, and (c) a primer P3comprising 3′-end portion Pa and a portion A1 that is different than B1and does not bind to the target nucleic acid. In one aspect, the abovereagents can be packaged in the same container. The kit can furtherinclude a reference nucleic acid and also may include a polymerase,nucleoside triphosphates, and a pair of primers for amplifying both thetarget nucleic acid and the reference nucleic acid sequences in order toincrease the number of molecules for the practice of the presentinvention particularly for detection of a mutation in a target nucleicacid sequence.

[0031] Another aspect of the present invention is a method for detectinga target nucleic acid sequence wherein a tailed target partial duplex A′is formed from the target sequence and is comprised of a duplex of saidtarget sequence, a label, and at one end of the duplex, twonon-complementary oligonucleotides, one linked to each strand. Acombination is provided comprising (i) the tailed target partial duplexA′ and (ii) a tailed reference partial duplex B′, which comprises aduplex of a sequence different than the target sequence, a label and, atone end of the duplex, two oligonucleotides that are complementary tothe two non-complementary oligonucleotides, one linked to each strand.The formation of a complex between the partial duplexes A′ and B′ isdetected by means of the labels, the formation thereof being directlyrelated to the presence of the target nucleic acid sequence.

[0032] Another embodiment of the present invention is a method ofdetecting a target nucleic acid sequence. The method comprisesamplification of the target sequence by polymerase chain reaction, usingprimers P1 and P2 to produce an amplicon AA. At least one of primers P1and P2 comprises a label and primer P1 is comprised of a 3′-end portionPa that can hybridize with the target sequence and 5′-end portion B1that cannot hybridize with the target sequence. A primer P3 is extendedby chain extension along one strand of amplicon AA to produce a tailedtarget partial duplex A′. Primer P3 is comprised of 3′-end portion Paand a 5′-end portion Al that cannot hybridize to the target sequence orits complement. A reference nucleic acid sequence different than thetarget nucleic acid sequence is amplified, using primer P2 and P3, bypolymerase chain reaction to produce amplicon BB. When primer P2 used inthe amplification of the target nucleic acid sequence comprises a label,primer P2 used for the amplification of the reference nucleic acidsequence comprises a label that may be the same or a different label.When primer P1 used in the amplification of the target nucleic acidsequence comprises a label, primer P3 comprises a label that may be thesame or a different label. Primer P1 is extended by chain extensionalong one strand of amplicon BB to produce a tailed reference partialduplex B′. The tailed target partial duplex A′ is allowed to bind to thetailed reference partial duplex B′ to form a complex. The binding of oneof the labels to another of the labels as a result of the formation ofthe complex is detected. Such binding is directly related to thepresence of the target nucleic acid sequence.

[0033] Another aspect of the present invention is a method for detectinga target nucleic acid sequence wherein a partial duplex A′ is producedfrom a target nucleic acid sequence. The duplex A′ comprises a fullycomplementary double stranded nucleic acid sequence containing thetarget nucleic acid sequence wherein one strand has at its 5′-end aportion A1 that does not hybridize with a corresponding portion A2 atthe 3′-end of the other strand. At least one of the strands of thepartial duplex A′ comprises a label. A partial duplex B′ is producedfrom a reference nucleic acid sequence and comprises a double strandednucleic acid sequence different from the target nucleic acid sequence.The strand corresponding to the strand comprising portion A1 has at its5′-end a portion B1 that is complementary with A2 and the other strandhas at its 3′-end a portion B2 that is complementary with Al. One of thestrands of partial duplex B′ comprises a label wherein such strand isunable to hybridize directly to the strand of partial duplex A′ thatcomprises a label. Partial duplexes A′ and B′ are subjected toconditions that permit the duplexes to hybridize to each other to form astable quadramolecular complex. A determination is made as to whethersuch complex is formed. The presence thereof indicates the presence ofthe target nucleic acid sequence. In an alternate embodiment the strandscomprising A1 and B1 have labels instead of the strands comprising A2and B2.

[0034] Another embodiment of the present invention is a method fordetecting the presence of a difference between two related nucleic acidsequences wherein a target nucleic acid sequence and a reference nucleicacid sequence are produced from the two related nucleic acid sequences.Each respective strand of the target nucleic acid sequence and thereference nucleic acid sequence produced has a portion introducedtherein that is a nucleotide sequence priming site. A partial duplex A′is produced from the target nucleic acid sequence using the nucleotidesequence priming sites. Partial duplex A′ comprises a fullycomplementary double stranded nucleic acid sequence containing thetarget nucleic acid sequence wherein one strand has at its 5′-end aportion Al that does not hybridize with a corresponding portion A2 atthe 3′-end of the other strand. One of the strands of the partial duplexA′ may comprise a label. A partial duplex B′ is also produced from thereference nucleic acid sequence using the nucleotide sequence primingsites. Partial duplex B′ comprises the double stranded nucleic acidsequence wherein the strand corresponding to the strand comprisingportion Al has at its 5′-end a portion B1 that is complementary with A2and the other strand has at its 3′-end a portion B2 that iscomplementary with A1. One of the strands of the partial duplex B′ maycomprise a label wherein a strand comprising a label is unable tohybridize directly to a strand of the partial duplex A′ when that strandcomprises a label. The partial duplexes A′ and B′ are subjected toconditions that permit the duplexes to hybridize to each other. If therelated nucleic acid sequences have a difference, a stable complex isformed comprising partial duplex A′ and partial duplex B′. Adetermination is made as to whether a stable complex is formed, thepresence thereof indicating the presence of a difference between the tworelated nucleic acid sequences.

[0035] Another embodiment of the present invention is a method fordetecting the presence of a mutation in a target nucleic acid sequencecomprising amplification of target and reference nucleic acid sequencesby polymerase chain reaction using primers PX1 i and PX2 i to produce atarget sequence or a reference sequence, respectively, comprisingnucleotide sequence priming sites Pa′ and P2′. The reference sequence isidentical to the target sequence but lacks a possible mutation. Thetarget sequence produced above is amplified by polymerase chainreaction, using primers P1 and P2, to produce an amplicon AA. One ofprimers P1 and P2 may comprise a label and primer P1 is comprised of a3′-end portion Pa that can hybridize with priming site Pa′ of the targetsequence and 5′-end portion B1 that cannot hybridize with the targetsequence. A primer P3 is extended by chain extension along one strand ofamplicon AA to produce a tailed target partial duplex A′. Primer P3 iscomprised of 3′-end portion Pa and a 5′-end portion A1 that cannothybridize to the target sequence or its complement. The referencesequence produced above is amplified, using primer P2 and primer P3, bypolymerase chain reaction to produce amplicon BB. Primer P2 may comprisea label when primer P2 above comprises a label and primer P3 maycomprise a label when primer P1 above comprises a label. Primer P1 isextended by chain extension along one strand of amplicon BB to produce atailed reference partial duplex B′. The tailed target partial duplex A′is allowed to bind to the tailed reference partial duplex B′. Theformation of a stable complex between the tailed partial duplexes isdetected, the formation thereof being directly related to the presenceof the mutation.

[0036] Another embodiment of the invention is a kit as mentioned abovethat also includes a pair of adapter primers for amplifying the targetand reference nucleic acids. One of the primers has a 3′-end portionthat is hybridizable to the target and reference nucleic acids and aportion 5′ thereof that is not hybridizable with the target or referencenucleic acids and is substantially identical to primer P2. The other ofthe primers has a 3′-end portion that is hybridizable to the target andreference nucleic acids and a portion 5′ thereof that is nothybridizable with the target or reference nucleic acids and issubstantially identical to the 3′-end portion Pa of primers P1 and P3.The adapter primers are usually packaged in a container separate fromprimers P1, P2 and P3.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGS. 1-11 are schematic diagrams depicting alternate embodimentsin accordance with the present invention.

[0038]FIGS. 1B and 2B are schematic diagrams/′ depicting embodimentswherein there is no difference between target and reference nucleic acidsequences.

[0039]FIG. 8B is a schematic diagram depicting an embodiment wherein notarget nucleic acid sequence is present in the sample.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0040] The present invention is universal and permits detection of anydifference in two related nucleic acid sequences, whether or not suchdifference is known. Such differences include any mutation includingsingle base substitution, deletion or insertion within a sequence thatcan be defined by a pair of primers for conducting the polymerase chainreaction. The method may be homogeneous or heterogeneous,non-radioactive, fast and amenable to automation. It is ideally suitedfor rapid mutation pre-screening. The invention also has application inthe area of amplification by polymerase chain reaction. The presentinvention permits PCR and subsequent steps, such as detection of the PCRproducts, to be conducted without the need for additional probes in asingle container without a separation step.

[0041] In one aspect the present method involves formation of afour-strand DNA structure or complex from DNA. The formation involvesproducing two partial duplexes by amplification by using three differentprimers in the polymerase chain reaction and allowing the amplifiedproducts to anneal. The complex dissociates into normal duplexstructures by strand exchange by means of branch migration when thehybridized portions of each partial duplex are identical. However, wherethere is a difference between the two hybridized portions, the complexdoes not dissociate and can be detected as an indication of the presencea difference between the nucleic acids. A particularly attractivefeature of the present invention is that the reactions may be carriedout simultaneously in the same medium without a separation step.

[0042] Before proceeding further with a description of the specificembodiments of the present invention, a number of terms will be defined.

[0043] Nucleic acid—a compound or composition that is a polymericnucleotide or polynucleotide. The nucleic acids include both nucleicacids and fragments thereof from any source, in purified or unpurifiedform including DNA (dsDNA and ssDNA) and RNA, including t-RNA, m-RNA,r-RNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RNAhybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomesof biological material such as microorganisms, e.g., bacteria, yeasts,viruses, viroids, molds, fungi, plants, animals, humans, and the like.The nucleic acid can be only a minor fraction of a complex mixture suchas a biological sample. The nucleic acid can be obtained from abiological sample by procedures well known in the art. Also included aregenes, such as hemoglobin gene for sickle-cell anemia, cystic fibrosisgene, oncogenes, cDNA, and the like. Where the nucleic acid is RNA, itis first converted to cDNA by means of a primer and reversetranscriptase. The nucleotide polymerase used in the present inventionfor carrying out amplification and chain extension can have reversetranscriptase activity. Sequences of interest may be embedded insequences of any length of the chromosome, cDNA, plasmid, etc.

[0044] Sample—the material suspected of containing the nucleic acid.Such samples include biological fluids such as blood, serum, plasma,sputum, lymphatic fluid, semen, vaginal mucus, feces, urine, spinalfluid, and the like; biological tissue such as hair and skin; and soforth. Other samples include cell cultures and the like, plants, food,forensic samples such as paper, fabrics and scrapings, water, sewage,medicinals, etc. When necessary, the sample may be pretreated withreagents to liquefy the sample and release the nucleic acids frombinding substances. Such pretreatments are well known in the art.

[0045] Amplification of nucleic acids—any method that results in theformation of one or more copies of a nucleic acid (exponentialamplification). One such method for enzymatic amplification of specificsequences of DNA is known as the polymerase chain reaction (PCR), asdescribed by Saiki, et al., supra. This in vitro amplification procedureis based on repeated cycles of denaturation, oligonucleotide primerannealing, and primer extension by thermophilic template dependentpolynucleotide polymerase, resulting in the exponential increase incopies of the desired sequence of the nucleic acid flanked by theprimers. The two different PCR primers are designed to anneal toopposite strands of the DNA at positions that allow the polymerasecatalyzed extension product of one primer to serve as a template strandfor the other, leading to the accumulation of a discrete double strandedfragment whose length is defined by the distance between the 5′ ends ofthe oligonucleotide primers. Primer length can vary from about 10 to 50or more nucleotides and are usually selected to be at least about 15nucleotides to ensure high specificity. The double stranded fragmentthat is produced is called an “amplicon” and may vary in length form asfew as about 30 nucleotides to 10,000 or more.

[0046] Chain extension of nucleic acids—extension of the 3′-end of apolynucleotide in which additional nucleotides or bases are appended.Chain extension relevant to the present invention is template dependent,that is, the appended nucleotides are determined by the sequence of atemplate nucleic acid to which the extending chain is hybridized. Thechain extension product sequence that is produced is complementary tothe template sequence. Usually, chain extension is enzyme catalyzed,preferably, in the present invention, by a thermophilic DNA polymerase.

[0047] Target nucleic acid sequence—a sequence of nucleotides to bestudied either for the presence of a difference from a related sequenceor for the determination of its presence or absence. The target nucleicacid sequence may be double stranded or single stranded and from anatural or synthetic source. When the target nucleic acid sequence issingle stranded, the method of the present invention produces a nucleicacid duplex comprising the single stranded target nucleic acid sequence.

[0048] The target sequence usually exists within a portion or all of anucleic acid, the identity of which is known to an extent sufficient toallow preparation of various primers necessary for introducing one ormore priming sites flanking the target sequence or conducting anamplification of the target sequence or a chain extension of theproducts of such amplification in accordance with the present invention.

[0049] Accordingly, other than for the sites to which the primers bind,the identity of the target nucleic acid sequence may or may not beknown. In general, in PCR, primers hybridize to, and are extended along(chain extended), at least the target sequence, and, thus, the targetsequence acts as a template. The target sequence usually contains fromabout 30 to 20,000 or more nucleotides, more frequently, 100 to 10,000nucleotides, preferably, 50 to 1,000 nucleotides. The target nucleicacid sequence is generally a fraction of a larger molecule or it may besubstantially the entire molecule. The minimum number of nucleotides inthe target sequence is selected to assure that a determination of adifference between two related nucleic acid sequences in accordance withthe present invention can be achieved.

[0050] Reference nucleic acid sequence —a nucleic acid sequence that isrelated to the target nucleic acid in that the two sequences areidentical except for the presence of a difference, such as a mutation.Where a mutation is to be detected, the reference nucleic acid sequenceusually contains the normal or “wild type” sequence. In certainsituations the reference nucleic acid sequence may be part of the sampleas, for example, in samples from tumors, the identification of partiallymutated microorganisms, or identification of heterozygous carriers of amutation. Consequently, both the reference and the target nucleic acidsequences are subjected to similar or the same amplification conditions.As with the target nucleic acid sequence, the identity of the referencenucleic acid sequence need be known only to an extent sufficient toallow preparation of various primers necessary for introducing one ormore priming sites flanking the reference sequence or conducting anamplification of the target sequence or a chain extension of theproducts of such amplification in accordance with the present invention.Accordingly, other that for the sites to which the primers bind, theidentity of the reference nucleic acid sequence may or may not be known.The reference nucleic acid sequence may be a reagent employed in themethods in accordance with the present invention. This is particularlythe situation where the present method is used in PCR amplification fordetection of a target nucleic acid sequence. Depending on the method ofpreparation of this reagent it may or may not be necessary to know theidentity of the reference nucleic acid. The reference nucleic acidreagent may be obtained form a natural source or prepared by knownmethods such as those described below in the definition ofoligonucleotides.

[0051] Holliday junction—the branch point in a four way junction in acomplex of two identical nucleic acid sequences and their complementarysequences. The junction is capable of undergoing branch migrationresulting in dissociation into two double stranded sequences wheresequence identity and complementarity extend to the ends of the strands.

[0052] Complex—a complex of four nucleic acid strands containing aHolliday junction, which is inhibited from dissociation into two doublestranded sequences because of a difference in the sequences and theircomplements. Accordingly, the complex is quadramolecular.

[0053] Related nucleic acid sequences—two nucleic acid sequences arerelated when they contain at least 15 nucleotides at each end that areidentical but have different lengths or have intervening sequences thatdiffer by at least one nucleotide. Frequently, related nucleic acidsequences differ from each other by a single nucleotide. Such differenceis referred to herein as the “difference between two related nucleicacid sequences.” A difference can be produced by the substitution,deletion or insertion of any single nucleotide or a series ofnucleotides within a sequence.

[0054] Mutation—a change in the sequence of nucleotides of a normallyconserved nucleic acid sequence resulting in the formation of a mutantas differentiated from the normal (unaltered) or wild type sequence.Mutations can generally be divided into two general classes, namely,base-pair substitutions and frameshift mutations. The latter entail theinsertion or deletion of one to several nucleotide pairs. A differenceof one nucleotide can be significant as to phenotypic normality orabnormality as in the case of, for example, sickle cell anemia.

[0055] Partial duplex—a fully complementary double stranded nucleic acidsequence wherein one end thereof has non-complementary oligonucleotidesequences, one linked to each strand of the double stranded molecule,each non-complementary sequence having 8 to 60, preferably, 10 to 50,more preferably, 15 to 40, nucleotides. Thus, the partial duplex is saidto be “ailed” because each strand of the duplex has a single strandedoligonucleotide chain linked thereto.

[0056] Duplex—a double stranded nucleic acid sequence wherein all of thenucleotides therein are complementary.

[0057] Oligonucleotide—a single stranded polynucleotide, usually asynthetic polynucleotide. The oligonucleotide(s) are usually comprisedof a sequence of 10 to 100 nucleotides, preferably, 20 to 80nucleotides, and more preferably, 30 to 60 nucleotides in length.

[0058] Various techniques can be employed for preparing anoligonucleotide utilized in the present invention. Such oligonucleotidecan be obtained by biological synthesis or by chemical synthesis. Forshort sequences (up to about 100 nucleotides) chemical synthesis willfrequently be more economical as compared to the biological synthesis.In addition to economy, chemical synthesis provides a convenient way ofincorporating low molecular weight compounds and/or modified basesduring the synthesis step. Furthermore, chemical synthesis is veryflexible in the choice of length and region of the target polynucleotidebinding sequence. The oligonucleotide can be synthesized by standardmethods such as those used in commercial automated nucleic acidsynthesizers. Chemical synthesis of DNA on a suitably modified glass orresin can result in DNA covalently attached to the surface. This mayoffer advantages in washing and sample handling. For longer sequencesstandard replication methods employed in molecular biology can be usedsuch as the use of M13 for single stranded DNA as described by J.Messing (1983) Methods Enzymol, 101, 20-78.

[0059] Other methods of oligonucleotide synthesis includephosphotriester and phosphodiester methods (Narang, et al. (1979) Meth.Enzymol 68: 90) and synthesis on a support (Beaucage, et al. (1981)Tetrahedron Letters 22: 1859-1862) as well as phosphoramidate technique,Caruthers, M. H., et al., “Methods in Enzymology,” Vol. 154, pp. 287-314(1988), and others described in “Synthesis and Applications of DNA andRNA,” S.A. Narang, editor, Academic Press, New York, 1987, and thereferences contained therein.

[0060] Oligonucleotide primer(s)—an oligonucleotide that is usuallyemployed in a chain extension on a polynucleotide template such as in,for example, an amplification of a nucleic acid. The oligonucleotideprimer is usually a synthetic oligonucleotide that is single stranded,containing a hybridizable sequence at its 3′-end that is capable ofhybridizing with a defined sequence of the target or referencepolynucleotide. Normally, the hybridizable sequence of theoligonucleotide primer has at least 90%, preferably 95%, most preferably100%, complementarity to a defined sequence or primer binding site. Thenumber of nucleotides in the hybridizable sequence of an oligonucleotideprimer should be such that stringency conditions used to hybridize theoligonucleotide primer will prevent excessive random non-specifichybridization. Usually, the number of nucleotides in the hybridizablesequence of the oligonucleotide primer will be at least ten nucleotides,preferably at least 15 nucleotides and, preferably 20 to 50,nucleotides. In addition, the primer may have a sequence at its 5′-endthat does not hybridize to the target or reference polynucleotides thatcan have 1 to 60 nucleotides, preferably, 8 to 30 polynucleotides.

[0061] Nucleoside triphosphates—nucleosides having a 5′-triphosphatesubstituent. The nucleosides are pentose sugar derivatives ofnitrogenous bases of either purine or pyrimidine derivation, covalentlybonded to the 1′-carbon of the pentose sugar, which is usually adeoxyribose or a ribose. The purine bases comprise adenine(A), guanine(G), inosine (I), and derivatives and analogs thereof. The pyrimidinebases comprise cytosine (C), thymine (T), uracil (U), and derivativesand analogs thereof. Nucleoside triphosphates includedeoxyribonucleoside triphosphates such as the four common triphosphatesdATP, dCTP, dGTP and dTTP and ribonucleoside triphosphates such as thefour common triphosphates rATP, rCTP, rGTP and rUTP.

[0062] The term “nucleoside triphosphates” also includes derivatives andanalogs thereof, which are exemplified by those derivatives that arerecognized and polymerized in a similar manner to the underivatizednucleoside triphosphates. Examples of such derivatives or analogs, byway of illustration and not limitation, are those which arebiotinylated, amine modified, alkylated, and the like and also includephosphorothioate, phosphite, ring atom modified derivatives, and thelike.

[0063] Nucleotide—a base-sugar-phosphate combination that is themonomeric unit of nucleic acid polymers, i.e., DNA and RNA.

[0064] Modified nucleotide—is the unit in a nucleic acid polymer thatresults from the incorporation of a modified nucleoside triphosphateduring an amplification reaction and therefore becomes part of thenucleic acid polymer.

[0065] Nucleoside—is a base-sugar combination or a nucleotide lacking aphosphate moiety.

[0066] Nucleotide polymerase—a catalyst, usually an enzyme, for formingan extension of a polynucleotide along a DNA or RNA template where theextension is complementary thereto. The nucleotide polymerase is atemplate dependent polynucleotide polymerase and utilizes nucleosidetriphosphates as building blocks for extending the 3′-end of apolynucleotide to provide a sequence complementary with thepolynucleotide template. Usually, the catalysts are enzymes, such as DNApolymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4DNA polymerase, T7 DNA polymerase, Klenow fragment, and reversetranscriptase, and are preferably thermally stable DNA polymerases suchas Vent® DNA polymerase, VentR® DNA polymerase, Pfu® DNA polymerase,Taq® DNA polymerase, and the like, derived from any source such ascells, bacteria, such as E. coli, plants, animals, virus, thermophilicbacteria, and so forth.

[0067] Wholly or partially sequentially—when the sample and variousagents utilized in the present invention are combined other thanconcomitantly (simultaneously), one or more may be combined with one ormore of the remaining agents to form a subcombination. Subcombinationand remaining agents can then be combined and can be subjected to thepresent method.

[0068] Hybridization (hybridizing) and binding—in the context ofnucleotide sequences these terms are used interchangeably herein. Theability of two nucleotide sequences to hybridize with each other isbased on the degree of complementarity of the two nucleotide sequences,which in turn is based on the fraction of matched complementarynucleotide pairs. The more nucleotides in a given sequence that arecomplementary to another sequence, the more stringent the conditions canbe for hybridization and the more specific will be the binding of thetwo sequences. Increased stringency is achieved by elevating thetemperature, increasing the ratio of cosolvents, lowering the saltconcentration, and the like.

[0069] Complementary—Two sequences are complementary when the sequenceof one can bind to the sequence of the other in an anti-parallel sensewherein the 3′-end of each sequence binds to the 5′-end of the othersequence and each A, T(U), G, and C of one sequence is then aligned witha T(U), A, C, and G, respectively, of the other sequence.

[0070] Copy—means a sequence that is a direct identical copy of a singlestranded polynucleotide sequence as differentiated from a sequence thatis complementary to the sequence of such single stranded polynucleotide.

[0071] Conditions for extending a primer—includes a nucleotidepolymerase, nucleoside triphosphates or analogs thereof capable ofacting as substrates for the polymerase and other materials andconditions required for enzyme activity such as a divalent metal ion(usually magnesium), pH, ionic strength, organic solvent (such asformamide), and the like.

[0072] Member of a specific binding pair (“sbp member”)—one of twodifferent molecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These may be members of an immunological pairsuch as antigen-antibody, or may be operator-repressor,nuclease-nucleotide, biotin-avidin, hormone-hormone receptor,IgG-protein A, DNA-DNA, DNA-RNA, and the like.

[0073] Ligand—any compound for which a receptor naturally exists or canbe prepared.

[0074] Receptor (“antiligand”)—any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring and synthetic receptors, e.g., thyroxine bindingglobulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids,repressors, oligonucleotides, protein A, complement component C1q, orDNA binding proteins and the like.

[0075] Small organic molecule—a compound of molecular weight less thanabout 1500, preferably 100 to 1000, more preferably 300 to 600 such asbiotin, digoxin, fluorescein, rhodamine and other dyes, tetracycline andother protein binding molecules, and haptens, etc. The small organicmolecule can provide a means for attachment of a nucleotide sequence toa label or to a support.

[0076] Support or surface—a porous or non-porous water insolublematerial. The support can be hydrophilic or capable of being renderedhydrophilic and includes inorganic powders such as silica, magnesiumsulfate, and alumina; natural polymeric materials, particularlycellulosic materials and materials derived from cellulose, such as fibercontaining papers, e.g., filter paper, chromatographic paper, etc.;synthetic or modified naturally occurring polymers, such asnitrocellulose, cellulose acetate, poly (vinyl chloride),polyacrylamide, cross linked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), etc.; either used by themselves or in conjunction with othermaterials; glass available as Bioglass, ceramics, metals, and the like.Natural or synthetic assemblies such as liposomes, phospholipidvesicles, and cells can also be employed.

[0077] Binding of sbp members to a support or surface may beaccomplished by well-known techniques, commonly available in theliterature. See, for example, “Immobilized Enzymes,” Ichiro Chibata,Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059(1970). The surface can have any one of a number of shapes, such asstrip, rod, particle, including bead, and the like.

[0078] Label—a member of a signal producing system. Labels includereporter molecules that can be detected directly by virtue of generatinga signal, and specific binding pair members that may be detectedindirectly by subsequent binding to a cognate that contains a reportermolecule such as oligonucleotide sequences that can serve to bind acomplementary sequence or a specific DNA binding protein; organicmolecules such as biotin or digoxigenin that can bind respectively tostreptavidin and antidigoxin antibodies, respectively; polypeptides;polysaccharides; and the like. In general, any reporter molecule that isdetectable can be used. The reporter molecule can be isotopic ornonisotopic, usually non-isotopic, and can be a catalyst, such as anenzyme, dye, fluorescent molecule, chemiluminescer, coenzyme, enzymesubstrate, radioactive group, a particle such as latex or carbonparticle, metal sol, crystallite, liposome, cell, etc., which may or maynot be further labeled with a dye, catalyst or other detectable group,and the like. The reporter group can be a fluorescent group such asfluorescein, a chemiluminescent group such as luminol, a terbiumchelator such as N-(hydroxyethyl) ethylenediaminetriacetic acid that iscapable of detection by delayed fluorescence, and the like.

[0079] The label is a member of a signal producing system and cangenerate a detectable signal either alone or together with other membersof the signal producing system. As mentioned above, a reporter moleculecan serve as a label and can be bound directly to a nucleotide sequence.Alternatively, the reporter molecule can bind to a nucleotide sequenceby being bound to an sbp member complementary to an sbp member thatcomprises a label bound to a nucleotide sequence. Examples of particularlabels or reporter molecules and their detection can be found in U.S.patent application Ser. No. 07/555,323 filed Jul. 19, 1990, the relevantdisclosure of which is incorporated herein by reference.

[0080] Signal Producing System—the signal producing system may have oneor more components, at least one component being the label. The signalproducing system generates a signal that relates to the presence of adifference between the target polynucleotide sequence and the referencepolynucleotide sequence. The signal producing system includes all of thereagents required to produce a measurable signal. When a reportermolecule is not conjugated to a nucleotide sequence, the reportermolecule is normally bound to an sbp member complementary to an sbpmember that is bound to or part of a nucleotide sequence. Othercomponents of the signal producing system can include substrates,enhancers, activators, chemiluminescent compounds, cofactors,inhibitors, scavengers, metal ions, specific binding substances requiredfor binding of signal generating substances, coenzymes, substances thatreact with enzymic products, enzymes and catalysts, and the like. Thesignal producing system provides a signal detectable by external means,such as by use of electromagnetic radiation, electrochemical detection,desirably by spectrophotometric detection. The signal-producing systemis described more fully in U.S. patent application Ser. No. 07/555,323,filed Jul. 19, 1990, the relevant disclosure of which is incorporatedherein by reference.

[0081] Ancillary Materials—Various ancillary materials will frequentlybe employed in the methods and assays carried out in accordance with thepresent invention. For example, buffers will normally be present in theassay medium, as well as stabilizers for the assay medium and the assaycomponents. Frequently, in addition to these additives, proteins may beincluded, such as albumins, organic solvents such as formamide,quaternary ammonium salts, polycations such as dextran sulfate,surfactants, particularly non-ionic surfactants, binding enhancers,e.g., polyalkylene glycols, or the like.

[0082] As mentioned above, one aspect of the present invention concernsa method for detecting the presence of a difference between two relatednucleic acid sequences. In the method, if there is a difference betweenthe two related nucleic acid sequences, a stable quadramolecular complexis formed comprising both of the nucleic acid sequences in doublestranded form. Usually, the complex comprises a Holliday junction. Bothmembers of at least one pair of non-complementary strands within thecomplex have labels. The association of the labels as part of thecomplex is determined as an indication of the presence of the differencebetween the two related sequences. The method may be employed fordetecting the presence of a mutation in a target nucleic acid sequenceor for detecting the presence of a target nucleic acid sequence.

[0083] One aspect of the invention is depicted in FIG. 1.Quadramolecular complex C comprises partial duplex A′ and partial duplexB′, Partial duplexes A′ and B′ are related in that their hybridizedportions are identical except for mutation M in partial duplex A′.Additionally, partial duplex A′ has a label L1, which may or may notdiffer from label L2 in partial duplex B′. Oligonucleotide tail A1 ofpartial duplex A′ is hybridized to corresponding oligonucleotide tail B2of partial duplex B′ and, similarly, oligonucleotide tail A2 of partialduplex A′ is hybridized to oligonucleotide tail B1 of partial duplex B′.Accordingly, complex C is quadramolecular and contains a four wayjunction H. Because oligonucleotide tails A1 and B1 are different,branch migration can only proceed away from these tails and then onlyuntil mutation M is reached, at which point branch migration stops.Thus, when a mutation is present, complex C is stable and can bedetected by determining whether both labels L1 and L2 have becomeassociated. The association of the labels indicates the presence ofcomplex C and thus the presence of mutation M in the target nucleic acidsequence. If mutation M is not present (see FIG. 1A), branch migrationcontinues until complete strand exchange occurs and only separateduplexes D and E are present whereupon no complex C is detected.

[0084] Another embodiment in accordance with the present invention isdepicted in FIG. 2. The method is for detecting a mutation within atarget nucleic acid sequence A that contains mutation M. The methodcomprises forming from the target sequence a tailed target partialduplex A′ comprised of a duplex of the target sequence, a label L1 andat one end of the duplex, two non-complementary oligonucleotides A1 andA2, one linked to each strand of duplex A′. Oligonucleotides A1 and A2have from 8 to 60 nucleotides, preferably, 15 to 30 nucleotides. Thetailed target partial duplex is provided in combination with a labeledtailed reference partial duplex B′ lacking mutation M. The tailedreference partial duplex B′ is comprised of two nucleic acid strandsthat are complementary to the strands in A′ but for mutation M.Accordingly, one terminus of the tailed reference partial duplex B′ has,as the end part of each strand, a sequence of nucleotides B1 and B2,respectively, that are complementary to A2 and A1, respectively, of A′and are not complementary to each other. Labels L1 and L2 are present innon-complementary strands of the tailed target and tailed referencepartial duplexes A′ and B′, respectively, where L1 and L2 may be thesame or different.

[0085] A complex C is formed as described above for FIG. 1.Oligonucleotide tail A1 of A′ is hybridized to correspondingoligonucleotide tail B2 of B′ and, similarly, oligonucleotide tail A2 ofA′ is hybridized to oligonucleotide tail B1 of B′. Becauseoligonucleotide tails A1 and B1 are different, branch migration can onlyproceed away form these tails and then only until mutation M is reached,at which point branch migration stops. Thus, when a mutation is present,complex C is stable and can be detected by determining whether bothlabels L1 and L2 have become associated. The association of the labelsindicates the presence of complex C. The formation of complex C isdirectly related to the presence of the mutation. If mutation M is notpresent in the nucleic acid (see FIG. 2A), branch migration continuesuntil complete strand exchange has occurred and only the separateduplexes D and E are present. In this event no complex C is detected.

[0086] Another aspect of the present invention is shown in FIG. 3, whichdepicts, by way of example and not limitation, the production of tailedtarget partial duplex A′ from target nucleic acid duplex A havingmutation M and the production of tailed reference partial duplex B′ fromreference nucleic acid duplex B. In the embodiment of FIG. 3, A isamplified by the polymerase chain reaction using primers P1 and P2 toproduce an amplicon AA. Primer P2 contains a label Li and primer P1 iscomprised of a 3′-end portion Pa that can hybridize with the targetsequence and 5′-end portion B1 that cannot hybridize with the targetsequence. The amplification is carried out in the presence of anucleotide polymerase and nucleoside triphosphates using temperaturecycling. Amplicon AA has two strands, a labeled strand derived fromprimer P2 and an unlabeled strand derived from primer P1. The unlabeledstrand has a 5′-end portion B1 of primer P1 and the labeled strand has acorresponding 3′-end portion A2, which is the complement of B1.

[0087] The above amplification is carried out by polymerase chainreaction (PCR) utilizing temperature cycling to achieve denaturation ofduplexes, oligonucleotide primer annealing, and primer extension bythermophilic template dependent nucleotide polymerase. In conducting PCRamplification of nucleic acids, the medium is cycled between two tothree temperatures. The temperatures for the present method for theamplification by PCR generally range from about 50° C. to 100° C., moreusually, from about 60° C. to 95° C. Relatively low temperatures of fromabout 50° C. to 80° C. are employed for the hybridization steps, whiledenaturation is carried out at a temperature of from about 80° C. to100° C. and extension is carried out at a temperature of from about 70°C. to 80° C., usually about 72° C. to 74° C . The amplification isconducted for a time sufficient to achieve a desired number of copiesfor an accurate determination of whether or not two related nucleicacids have a difference. Generally, the time period for conducting themethod is from about 10 seconds to 10 minutes per cycle and any numberof cycles can be used from 1 to as high as 60 or more, usually 10 to 50,frequently, 20 to 45. As a matter of convenience it is usually desirableto minimize the time period and the number of cycles. In general, thetime period for a given degree of amplification can be minimized, forexample, by selecting concentrations of nucleoside triphosphatessufficient to saturate the polynucleotide polymerase, by increasing theconcentrations of polynucleotide polymerase and polynucleotide primer,and by using a reaction container that provides for rapid thermalequilibration. Generally, the time period for conducting theamplification in the method of the invention is from about 5 to 200minutes.

[0088] In an example of a typical temperature cycling as may beemployed, the medium is subjected to multiple temperature cycles ofheating at 90° C. to 100° C. for 10 seconds to 3 minutes and cooling to65° C. to 80° C. for a period of 10 seconds to 3 minutes.

[0089] Referring again to FIG. 3, a chain extension of primer P3 alongthe labeled strand of amplicon AA is then carried out to produce tailedtarget partial duplex A′. Primer P3 is comprised of a 3′-end portion Pa,which is identical to Pa of primer P1 and which binds to the labeledstrand of AA. P3 has 5′-end portion A1 that is not complementary toamplicon AA. The chain extension is carried out in the presence of anucleotide polymerase and nucleoside triphosphates under appropriatetemperature conditions so that only the complementary strand of thelabeled strand is produced and not a copy. In this particular embodimentthis is achieved by removing primers P2 and P1 prior to extension of P3in a manner as described hereinbelow. The complementary unlabeled strandof tailed target partial duplex A′ has a 5′-end portion A1, which is notcomplementary to the 3′-end portion A2 of the labeled strand of A′.Unless the PCR reaction is carried out to produce an excess of thelabeled strand, there will also be present the unlabeled strand from theamplification. This strand is not a template during chain extension toform partial duplex A′.

[0090] The conditions for carrying out the chain extension in accordancewith the present invention are similar to those for the amplificationdescribed above. In general, the medium is heated to a temperature of90° C. to 100° C. for a period of 5 to 500 seconds and then cooled to20° C. to 80° C. for a period of 5 to 2000 seconds followed by heatingto 40° C. to 80° C. for a period of 5 to 2000 seconds. Preferably, themedium is subjected to heating at 90° C. to 100° C. for a period of 10seconds to 3 minutes, cooling to 50° C. to 65° C. for a period of 10seconds to 2 minutes and heating to 70° C. to 80° C. for a period of 30seconds to 5 minutes.

[0091] In carrying out the present method, an aqueous medium isemployed. Other polar cosolvents may also be employed, usuallyoxygenated organic solvents of from 1-6, more usually from 1-4, carbonatoms, including alcohols, ethers and the like. Usually thesecosolvents, if used, are present in less than about 70 weight percent,more usually in less than about 30 weight percent.

[0092] The pH for the medium is usually in the range of about 4.5 to9.5, more usually in the range of about 5.5-8.5, and preferably in therange of about 6-8, usually about 8. In general for amplification, thepH and temperature are chosen and varied, as the case may be, so as tocause, either simultaneously or sequentially, dissociation of anyinternally hybridized sequences, hybridization of the oligonucleotideprimer with the target nucleic acid sequence, extension of the primer,and dissociation of the extended primer. Various buffers may be used toachieve the desired pH and maintain the pH during the determination.Illustrative buffers include borate, phosphate, carbonate, Tris,barbital and the like. The particular buffer employed is not critical tothis invention but in individual methods one buffer may be preferredover another. The buffer employed in the present methods normallycontains magnesium ion (Mg²⁺), which is commonly used with many knownpolymerases, although other metal ions such as manganese have also beenused. Preferably, magnesium ion is used at a concentration of from about1 to 20 mM, preferably, from about 1.5 to 10 mM, more preferably, 3-4mM. The magnesium can be provided as a salt, for example, magnesiumchloride and the like. The primary consideration is that the metal ionpermit the distinction between different nucleic acids in accordancewith the present invention.

[0093] The concentration of the nucleotide polymerase is usuallydetermined empirically. Preferably, a concentration is used that issufficient such that further increase in the concentration does notdecrease the time for the amplification by over 5-fold, preferably2-fold. The primary limiting factor generally is the cost of thereagent.

[0094] The amount of the target nucleic acid sequences that is to beexamined in accordance with the present invention can be as low as oneor two molecules in a sample. The priming specificity of the primersused for the detection of a difference between two related nucleic acidsand other factors will be considered with regard to the need to conductan initial amplification of the target nucleic acid. It is within thepurview of the present invention for detection of a mutation to carryout a preliminary amplification reaction to increase, by a factor of 10²or more, the number of molecules of the target nucleic acid sequence.The amplification can be by any convenient method such as PCR,amplification by single primer, NASBA, and so forth, but will preferablybe by PCR. A PCR amplification is depicted in FIG. 4. A primer PX1 thatbinds upstream of the P1 and P3 binding site, depicted as Pa′, andunlabeled primer P2 are utilized along with other reagents employed inaccordance with known PCR amplification technology. The primers annealto the appropriate strands of the target nucleic acid sequence TS andare extended along such strands to produce multiple copies of A.Alternatively, the number of molecules of the target nucleic acidsequence can be increased by PCR using primers P1 and P2 only or PX1 andPX2 only.

[0095] The amount of the target nucleic acid sequence to be subjected tosubsequent amplification using primers in accordance with the presentinvention may vary from about 1 to 10¹⁰, more usually from about 10³ to10⁸ molecules, preferably at least 10⁻²¹ M in the medium and may be10⁻¹⁰ to 10⁻¹⁹M, more usually 10⁻¹⁴ to 10⁻¹⁹M.

[0096] If an initial amplification of the target nucleic acid sequenceis carried to increase the number of molecules, it may be desirable, butnot necessary, to remove, destroy or inactivate the primers used in theinitial amplification depending on the nature of the protocol utilized.Accordingly, when the present method is carried out using step-wiseaddition of reagents for each separate reaction, such as, for example,in the embodiment of FIG. 3, primer P1 should be removed prior to theextension of primer P3. On the other hand, for example, in theembodiment described hereinbelow where the reactions are carried outsimultaneously, it is not necessary to remove any of the primers. Anexample, by way of illustration and not limitation, of an approach todestroy the primers is to employ an enzyme that can digest only singlestranded DNA. For example, an enzyme may be employed that has both 5′ to3′ and 3′ to 5′ exonuclease activities, such as, e.g., exo VII. Themedium is incubated at a temperature and for a period of time sufficientto digest the primers. Usually, incubation at 20° C. to 40° C. for aperiod of 10 to 60 minutes is sufficient for an enzyme having the aboveactivity. The medium is next treated to inactivate the enzyme, which canbe accomplished, for example, by heating for a period of time sufficientto achieve inactivation. Inactivation of the enzyme can be realizedusually upon heating the medium at 90° C. to 100° C. for 0.5 to 30minutes. Other methods of removing the primers will be suggested tothose skilled in the art. It has been found, however, that removal ofsuch primers is not necessary in carrying out the methods of theinvention.

[0097] The amount of the oligonucleotide primer(s) used in theamplification reaction in the present invention will be at least asgreat as the number of copies desired and will usually be 10⁻⁹ to 10⁻³M, preferably, 10⁻⁷ to 10⁻⁴ M. Preferably, the concentration of theoligonucleotide primer(s) is substantially in excess over, preferably atleast 100 times greater than, more preferably, at least 1000 timesgreater than, the concentration of the target nucleic acid sequence. Theconcentration of the nucleoside triphosphates in the medium can varywidely; preferably, these reagents are present in an excess amount forboth amplification and chain extension. The nucleoside triphosphates areusually present in 10⁻⁶ to 10⁻²M, preferably 10⁻⁵ to 10⁻³M.

[0098] The order of combining the various reagents may vary. The targetnucleic acid may be combined with a pre-prepared combination of primersPX1, unlabeled P2, labeled P2, and P1, nucleoside triphosphates andnucleotide polymerase. Alternatively, the target nucleic acid, forexample, can be combined with only primers PX1 and unlabeled P2 togetherwith the nucleoside triphosphates and polymerase. After temperaturecycling is carried out, the reaction mixture can be combined with theremaining primers P1 and labeled P2.

[0099] In the embodiment of FIG. 3, reference nucleic acid sequence B isin a separate medium; primer P2 and primer P3 are employed in apolymerase chain reaction to produce amplicon BB. The amplification iscarried out using temperature cycling under the conditions describedabove in the presence of a nucleotide polymerase and nucleosidetriphosphates. B is comprised of a sequence identical to A except formutation M. Generally, primer P2 used for this amplification contains alabel L2 that may be the same as or different than L1. Amplicon BB hastwo strands, a labeled strand derived from primer P2 and an unlabeledstrand derived from primer P3. The unlabeled strand has end portion A1of primer P3 and the labeled strand has corresponding end portion B2,which is the complement of A1.

[0100] A chain extension of primer P1 along the labeled strand ofamplicon BB is carried out, under the conditions mentioned above for thechain extension of primer P3 along the labeled strand in duplex AA, toproduce tailed reference partial duplex B′. As mentioned above, primerP1 is comprised of portion Pa, which binds to the labeled strand of BBand portion B1 that does not bind to amplicon BB. The chain extension iscarried out in the presence of a nucleotide polymerase and nucleosidetriphosphates under appropriate temperature conditions so that only thecomplement of the labeled strand is produced and not a copy. Theextended primer P1 has a 5′-end portion B1, which is not complementaryto end portion B2 of the labeled strand of B′. As can be seen, A′ and B′are related in that each of their labeled strands is complementary,except for mutation M, to the unlabeled strand of the other.

[0101] The strands of partial duplexes A′ and B′ are allowed to bind andundergo branch migration by combining the mixtures containing partialduplexes A′ and B′ and incubating the combination at a temperature of30° C. to 75° C., preferably 60° C. to 70° C., for at least one minute,preferably, 20 to 40 minutes, wherein complex C is formed as describedabove for FIGS. 1 and 2. Oligonucleotide tail A1 of A′ is hybridized tocorresponding oligonucleotide tail B2 of B′ and, similarly,oligonucleotide tail A2 of A′ is hybridized to oligonucleotide tail B1of B′. Branch migration within complex C continues under the abovetemperature conditions with separation of the complex into duplexes Dand E unless a mutation M is present, whereupon branch migration andstrand dissociation is inhibited. Complex C is then detected, thepresence of which is directly related to the presence of mutation M.

[0102] In the embodiment depicted in FIG. 3, labels L1 and L2 areincorporated into the partial duplexes that comprise complex C andprovide a means for detection of complex C. This is by way ofillustration and not limitation and other convenient methods fordetecting complex C may be employed, such as the use of a receptor forthe complex. In this approach there is required only one label, L1 orL2, which comprises an sbp member or a reporter molecule. A receptor forthe sbp member and a receptor that can bind to complex C by virtue of afeature other than L1 or L2 can both bind to complex C and provide ameans for detection.

[0103] In the embodiment of FIG. 3, the reactions are carried outindependently to produce tailed partial duplexes A′ and B′,respectively. Then, the reaction mixtures can be combined to allow therespective strands of A′ and B′ to bind to one another to form complexC.

[0104] Surprisingly, however, it was discovered that the reactions ofthe present invention can be carried out in the same reaction medium andmany or all of the reactions may be carried out simultaneously. This isa particularly attractive feature of the present invention. In thisapproach a combination is provided in a single medium. The combinationcomprises (i) a sample containing a target nucleic acid sequencesuspected of having a mutation, (ii) a reference nucleic acid sequence,which may be added separately if it is not known to be present in thesample and which corresponds to the target nucleic acid lacking themutation, which as explained above may be the wild type nucleic acid,(iii) a nucleotide polymerase, (iv) nucleoside triphosphates, and (v)primers P1, P2 and P3, wherein P2 may include primer P2 labeled with L1and primer P2 labeled with L2, or P2 may be unlabeled and primers P1 andP3 may be labeled respectively with L1 and L2. The medium is thensubjected to multiple temperature cycles of heating and cooling tosimultaneously achieve all of the amplification and chain extensionreactions described above for FIG. 3 except that in this embodimentthere is no need to avoid making copies of any of the extended primers.Preferably, in this embodiment, each cycle includes heating the mediumat 90° C. to 100° C. for 10 seconds to 3 minutes, cooling the medium to60° C. to 70° C. for a period of 10 seconds to 3 minutes, and heatingthe medium at 70° C. to 75° C. for a period of 10 seconds to 3 minutesalthough different temperatures may be required depending on the lengthsof the primer sequences. Following the above temperature cycling themedium is subjected to heating for a period of time sufficient todenature double stranded molecules, preferably, at 90° C. to 99° C. for10 seconds to 2 minutes, and cooled to 40° C. to 80° C., preferably 60°C. to 70° C., and held at this temperature for at least one minute,preferably for 20 minutes to 2 hours.

[0105] Following cooling of the medium all possible partial and completeduplexes are formed that can form from 1) single strands that have anycombination of reference or mutant sequences and 5′-ends A2 and B2, and2) single strands having any combination of reference or mutantsequences and 5′-ends A1 or B1 wherein the strands may further belabeled with either L1 or L2 when L1 and L2 are different. Among thepartial duplexes that are formed are the tailed partial duplexes A′ andB′, which can bind to each other to form complex C, which does notdissociate into duplexes D and E when a mutation is present. Adetermination of the presence of such a complex is then made toestablish the presence of a mutation in the target nucleic acidsequence. When primers P1 and P3 are labeled instead of primer P2, thelabels L1 and L2 in partial duplexes A′ and B′ are attached to tails A1and B1, respectively, which still provides for detection of complex Cwhen a mutation is present.

[0106] While all the steps of this determination are preferably carriedout in the same medium as that used for the above reactions, some or allof the steps can be carried out wholly or partially sequentially indifferent media. Thus, for example, PCR amplification of target sequenceA and target sequence B, each using primers P1, P2 and P3, can beconducted in separate solutions. The solution can then be combined,heated to 90° C. to 100° C. to denature strands and then incubated asbefore at 40° C. to 80° C. to permit formation of duplexes and complex Cwhen a mutation is present. Detection of complex C can then be carriedout directly in the combined solutions or by adding reagents requiredfor detection or by separating the complex C, for example, on a solidsurface, and detecting its presence on the surface.

[0107] When a single reaction medium is used for detecting a differencebetween a target and reference nucleic acid, it may be necessary toconduct an initial amplification to increase the concentration of thetarget nucleic acid molecules and reference nucleic acid moleculesrelative to that of other nucleic acids that may be present in thesample. To this end such initial amplification can be carried out usingtwo additional primers PX1 and PX2 that bind to sites on the target andreference nucleic acids, which sites are upstream of the P2 binding siteand the P1 and P3 binding site, respectively. This initial amplificationcan be carried out in the same medium as the above reactions. Thus,primers PX1, PX2, P1, P2 and P3 may all be combined with the target andreference sequences prior to temperature cycling. This is more readilyseen in FIG. 5, which depicts the initial amplification for a mutant DNAanalyte TS. Two primers PX1 and PX2 are employed and bind to sites on TSthat are upstream of the sites to which primers P1 and P2, respectively,bind. These sites are indicated by Pa′ and P2′, respectively, in FIG. 5.The sites to which primers PX1 and PX2 bind are generally within about 0to 500 nucleotides, preferably, about 0 to 200 nucleotides away from Pa′and P2′ and may overlap partially or completely with Pa′ and P2′. PX1and PX2 are extended along their respective strands. The amplificationproduces multiple copies of target nucleic acid sequence A. Afterappropriate denaturing, primers P1 and P2 are allowed to anneal to andextend along the respective strands of A to produce multiple copies ofAA. The above also occurs for the reference DNA to produce multiplecopies of reference nucleic acid B, which is further amplified withprimers P2 and P3 to produce multiple copies of BB.

[0108] Preferably, when an initial amplification using primers PX1 andPX2 is carried out, these primers will be designed to anneal to thetarget and the reference nucleic acids at a higher temperature than thatfor primers P1, P2 and P3, respectively. This is usually achieved byselecting PX1 and PX2 sequences that are longer or more GC rich than P2and the Pa binding sequence in P1 and P3. The initial amplification isthen carried out at temperatures that exceed the temperature requiredfor binding P1, P2 and P3 and the subsequent amplifications to form AAand BB are carried out at lower temperatures that permit P1. P2 and P3to bind. It is then possible to detect the difference between target andreference nucleic acid sequences by combining the sequences, primersPX1, PX2, P1, P2 and P3 wherein P2 or P1 and P3 are labeled,polynucleotide polymerase, nucleotides triphosphates, and optionally thereagents needed to detect complex C, all in one medium. The initialamplification is carried out at temperatures that permit PX1 and PX2,but not P1, P2 and P3, to bind to the target sequence whereuponsequences A and B are formed. Temperature cycling is then carried out ata lower temperature where P1, P2 and P3 can bind and be extended. Themixture is then heated to 90° C. to 100° C. to denature the duplexes andcooled to permit formation of partial duplexes AA and BB and theirhybridization to form complex C. The complex can then be detecteddirectly if all of the necessary reagents are present or detection canbe carried out in a separate step. The nature of primers PX1 and PX2, aswell as the appropriate temperature for binding of these primers to thetarget sequence, are generally determined empirically with reference tothe nucleotide composition of primers P1, P2 and P3.

[0109] In another approach in accordance with the present invention,priming sites for primers P1, P2 and P3 may be introduced to the targetand reference sequences, usually flanking the target or referencesequence. A PCR step is employed utilizing adapter primers consisting oftwo regions: a 3′-proximal region which is hybridizable to a particularpriming site on the target or reference nucleic acid sequence and a5′-proximal region which is not hybridizable to the target or referencenucleic acid sequence and has substantially the same sequence as the3′-proximal region of a primer used in amplifications described aboveemployed in the detection of differences between two related nucleicacids. By “substantially the same sequence” is meant that an extensionproduct produced in an amplification using the adapter primers containsa priming site to which such primer used in amplifications describedabove employed in the detection of differences between two relatednucleic acids can hybridize. Such adapter primers are used to preparetarget and reference nucleic acid sequences having specific, universalpriming sites incorporated therein, which in turn are used as templatesfor a universal set of primers used in the amplifications describedabove in accordance with the present method for detection of differencesbetween two related nucleic acid sequences.

[0110] To this end an amplification is conducted, prior toamplifications to form AA and BB, using two additional primers PX1 i andPX2 i that bind to sites on the target and reference nucleic acids. Thisamplification may be carried out in the same or different reactioncontainers or different reaction media from that in which theamplifications to form AA and BB are carried out. For example, primersPX1 i and PX2 i are combined with the target and reference sequences,either in the same or different reaction medium, and subjected totemperature cycling. This approach is depicted in FIG. 9, which show aninitial amplification for a mutant DNA analyte TS and a correspondingreference nucleic acid RS. Two primers PX1 i and PX2 i are employed andbind to respective priming sites on TS and RS. PX1 i has a 3′-endportion that can hybridize with the target and reference sequence and5′-end portion Pa that cannot hybridize with the target or referencesequence. PX2 i has a 3′-end portion that can hybridize with the targetand reference sequence and 5′-end portion P2 that cannot hybridize withthe target or reference sequence. PX1 i and PX2 i are extended alongtheir respective strands. The amplification produces multiple copies ofextended primers that comprise the relevant portion of the targetnucleic acid sequence and reference nucleic acid sequence flanked bypriming sites Pa and P2, designated A and B, respectively, in FIG. 9.

[0111] The reaction products from this initial amplification arecombined with primers P1, P2 and P3 as shown in FIG. 3. Primers P1 andP2 anneal to and extend along the respective strands of A to producemultiple copies of AA. The above also occurs for the reference DNA toproduce multiple copies of reference nucleic acid B, which is furtheramplified with primers P2 and P3 to produce multiple copies of BB. Theremainder of the reactions that occur are as described above to give A′and B′, which then can form complex C.

[0112] The embodiment of FIG. 9 permits the use of universal primers P1,P2 and P3. This means that one set of primers for carrying out thereactions to produce complex C can be used for the analysis of a largenumber of target nucleic acid sequences and corresponding referencenucleic acid sequences. Such an approach involves the use of primers PX1i and PX2 i, which are designed to introduce to the target and referencesequences priming sites for universal primers P1, P2 and P3. Therelationship of PX1 i and PX2 i are such that each contains a 5′-endportion that corresponds to the priming sequence portion, i.e., theportion of the target sequence to which the primer hybridizes, at the3′-end of primers P1, P2 or P3 as the case may be. In the embodimentshown in FIG. 9, PX1 i contains 5′-end portion P2, which results in theintroduction of priming site P2′ in TS to which P2 can hybridize. PrimerPX2 i contains 5′-end portion Pa, which results in the introduction ofpriming site Pa′ in TS to which Pa of primers P1 and P3 can hybridize.

[0113] It is within the purview of the present invention to utilize, inconjunction with the embodiment of FIG. 9, an initial amplification asdescribed above and exemplified in FIG. 5 to increase the concentrationof the target nucleic acid molecules and reference nucleic acidmolecules relative to that of other nucleic acids that may be present inthe sample.

[0114] The use of universal primers allows the methods in accordancewith the present invention to be carried out less expensively in someapplications than a method using a different set of such primers foreach target nucleic acid sequence to be analyzed. The approach hasparticular application in searching large, continuous stretches (tens orhundreds of kilobases) of genomic DNA for a single meaningful sequencealteration that may or may not be present. Such areas include thecomparison of DNA fragments in the neighborhood of a suspected gene inboth healthy and affected individuals, development of polymorphicmarkers for the construction of high resolution genetic maps, researchapplications for correlation of particular phenotypes in various modelorganisms with specific DNA alterations, studies of diversity within aspecies, and so forth.

[0115] As mentioned above, the identity of the target nucleic acidsequence does not need to be known except to the extent to allowpreparation of the necessary primers for carrying out the abovereactions. The present invention permits the determination of thepresence or absence of a mutation in a nucleic acid in a sample withoutthe need to fully identify the sequence of the nucleic acid.Accordingly, one is able to determine the presence of a mutation in anucleic acid between two sequences of nucleotides for which primers canbe made.

[0116] In the present invention one means of detecting thequadramolecular complex involves the use of two labels onnon-complementary strands. The labels become associated by virtue ofboth being present in the quadramolecular complex if a difference ispresent between the related sequences. Detection of the two labels inthe complex provides for detection of the complex. Generally, theassociation of the labels within the complex is detected. Thisassociation may be detected in many ways. For example, one of the labelscan be an sbp member and a complementary sbp member is provided attachedto a support. Upon the binding of the complementary sbp members to oneanother, the complex becomes bound to the support and is separated fromthe reaction medium. The other label employed is a reporter moleculethat is then detected on the support. The presence of the reportermolecule on the support indicates the presence of the complex on thesupport, which in turn indicates the presence of the mutation in thetarget nucleic acid sequence. An example of a system as described aboveis the enzyme-linked immunosorbent assay (ELISA), a description of whichis found in “Enzyme-Immunoassay,” Edward T. Maggio, editor, CRC Press,Inc., Boca Raton, Fla. (1980) wherein, for example, the sbp member isbiotin, the complementary sbp member is streptavidin and the reportermolecule is an enzyme such as alkaline phosphatase.

[0117] Detection of the signal will depend upon the nature of the signalproducing system utilized. If the reporter molecule is an enzyme,additional members of the signal producing system would include enzymesubstrates and so forth. The product of the enzyme reaction ispreferably a luminescent product, or a fluorescent or non-fluorescentdye, any of which can be detected spectrophotometrically, or a productthat can be detected by other spectrometric or electrometric means. Ifthe reporter molecule is a fluorescent molecule, the medium can beirradiated and the fluorescence determined. Where the label is aradioactive group, the medium can be counted to determine theradioactive count.

[0118] The association of the labels within the complex may also bedetermined by using labels that provide a signal only if the labelsbecome part of the complex. This approach is particularly attractivewhen it is desired to conduct the present invention in a homogeneousmanner. Such systems include enzyme channeling immunoassay, fluorescenceenergy transfer immunoassay, electrochemiluminescence assay, inducedluminescence assay, latex agglutination and the like.

[0119] In one aspect of the present invention detection of the complexis accomplished by employing at least one suspendable particle as asupport, which may be bound directly to a nucleic acid strand or may bebound to an sbp member that is complementary to an sbp member attachedto a nucleic acid strand. Such a particle serves as a means ofsegregating the bound target polynucleotide sequence from the bulksolution, for example, by seftling, electrophoretic separation ormagnetic separation. A second label, which becomes part of the complexif a mutation is present, is a part of the signal producing system thatis separated or concentrated in a small region of the solution tofacilitate detection. Typical labels that may be used in this particularembodiment are fluorescent labels, particles containing a sensitizer anda chemiluminescent olefin (see U.S. Ser. No. 07/923,069 filed Jul. 31,1992, the disclosure of which is incorporated herein by reference),chemiluminescent and electroluminescent labels.

[0120] Preferably, the particle itself can serve as part of a signalproducing system that can function without separation or segregation.The second label is also part of the signal producing system and canproduce a signal in concert with the particle to provide a homogeneousassay detection method. A variety of combinations of labels can be usedfor this purpose. When all the reagents are added at the beginning ofthe reaction, the labels are limited to those that are stable to theelevated temperatures used for amplification, chain extension, andbranch migration. In that regard it is desirable to employ as labelspolynucleotide or polynucleotide analogs having 5 to 20 or morenucleotides depending on the nucleotides used and the nature of theanalog. Polynucleotide analogs include structures such aspolyribonucleotides, polynucleoside phosphonates, peptido-nucleic acids,polynucleoside phosphorothioates, homo DNA and the like. In general,unchanged nucleic acid analogs provide stronger binding and shortersequences can be used. Included in the reaction medium areoligonucleotide or polynucleotide analogs that have sequences ofnucleotides that are complementary. One of these oligonucleotidesoligonucleotide analogs is attached to, for example, a reporter moleculeor a particle. The other is attached to a primer, either primer P2 orprimer P1 and/or P3 as a label. Neither the oligonucieotide norpolynucleotide analog should serve as a polynucleotide polymerasetemplate. This is achieved by using either a polynucleotide analog or apolynucleotide that is connected to the primer by an abasic group. Theabasic group comprises a chain of 1 to 20 or more atoms, preferably atleast 6 atoms, more preferably, 6 to 12 atoms such as, for example,carbon, hydrogen, nitrogen, oxygen, sulfur, and phosphorus, which may bepresent as various groups such as polymethylenes, polymethylene ethers,hydroxylated polymethylenes, and so forth. The abasic group convenientlymay be introduced into the primer during solid phase synthesis bystandard methods.

[0121] Under the proper annealing temperature an oligonucleotide orpolynucleotide analog attached to a reporter molecule or particle canbind to its complementary polynucleotide analog or oligonucleotideseparated by an abasic site that has become incorporated into partialduplexes A′ and B′ as labels during amplification. If the partialduplexes become part of a quadramolecular complex, the reporter moleculeor particle becomes part of the complex. By using differentpolynucleotide analogs or oligonucleotide sequences for labels, L1 andL2, two different reporter molecules or particles can become part of thecomplex. Various combinations of particles and reporter molecules can beused.

[0122] The particles, for example, may be simple latex particles or maybe particles comprising a sensitizer, chemiluminescer, fluorescer, dye,and the like. Typical particle/reporter molecule pairs include a dyecrystallite and a fluorescent label where binding causes fluorescencequenching or a tritiated reporter molecule and a particle containing ascintillator. Typical reporter molecule pairs include a fluorescentenergy donor and a fluorescent acceptor dye. Typical particle pairsinclude (1) two latex particles, the association of which is detected bylight scattering or turbidimetry, (2) one particle capable of absorbinglight and a second label particle which fluoresces upon accepting energyfrom the first, and (3) one particle incorporating a sensitizer and asecond particle incorporating a chemiluminescer as described for theinduced luminescence immunoassay referred to in U.S. Ser. No.07/704,569, filed May 22, 1991, entitled “Assay Method Utilizing InducedLuminescence”, which disclosure is incorporated herein by reference.

[0123] Briefly, detection of the quadramolecular complex using theinduced luminescence assay as applied in the present invention involvesemploying a photosensitizer as part of one label and a chemiluminescentcompound as part of the other label. If the complex is present thephotosensitizer and the chemiluminescent compound come into closeproximity. The photosensitizer generates singlet oxygen and activatesthe chemiluminescent compound when the two labels are in closeproximity. The activated chemiluminescent compound subsequently produceslight. The amount of light produced is related to the amount of thecomplex formed.

[0124] By way of illustration as applied to the present invention, aparticle is employed, which comprises the chemiluminescent compoundassociated therewith such as by incorporation therein or attachmentthereto. The particles have a recognition sequence, usually anoligonucleotide or polynucleotide analog, attached thereto with acomplementary sequence incorporated into one of the nucleic acid strandsas a label, L1. Another particle is employed that has thephotosensitizer associated therewith. These particles have a recognitionsequence attached thereto, which is different than that attached to thechemiluminescent particles. A complementary sequence is incorporated asa label L2 in the nucleic acid strand in complex C that is notcomplementary to the nucleic acid strand carrying label L1. Once themedium has been treated in accordance with the present invention to forma quadramolecular complex C, the medium is irradiated with light toexcite the photosensitizer, which is capable in its excited state ofactivating oxygen to a singlet state. Because the chemiluminescentcompound of one of the sets of particles is now in close proximity tothe photosensitizer by virtue of the presence of the targetpolynucleotide having a mutation, the chemiluminescent compound isactivated by the singlet oxygen and emits luminescence. The medium isthen examined for the presence and/or the amount of luminescence orlight emitted, the presence thereof being related to the presence ofquadramolecular complex C. The presence of the latter indicates thepresence and/or amount of the target polynucleotide having a mutation orof the target polynucleotide itself.

[0125] Another aspect of the present invention is depicted in FIG. 6. Amethod is shown for preparing a DNA partial duplex having a portion atan end thereof that has two predefined non-complementary single strandedsequences B1 and B2. A medium containing a double stranded nucleic acidB is combined with a polymerase, nucleoside triphosphates and twoprimers, P3 and P2. P3 is extendable along one of the strands of thenucleic acid. P3 has a 3′-end portion Pa that does bind to this strandand a 5′-end portion A1 that does not bind thereto. P2 is extendablealong the other of the strands of the nucleic acid. Extended primerproduced by the extension of one of the primers is a template for theother of the primers. The medium is subjected to temperature cycling toextend the primers. As a result duplex BB is produced. The conditionsfor carrying out this step are the same as those described above foramplification of a nucleic acid. The medium is then combined with aprimer P1, which has 3′-end portion Pa and a 5′-end portion Bl that doesnot bind to the extended primers of duplex BB. The medium is nextsubjected to conditions such that P1 binds to and is extended alongextended primer P2 to produce only a complement, and not a copy, of theextended primer. The conditions employed are as described above forchain extension of a primer only, not as part of an amplification. Apartial duplex B′ is formed that contains non-complementary end portionsB1 and B2. These end portions are predefined by virtue of the primersemployed in the reaction permitting one to introduce desirednon-complementary sequences at the end of a double stranded nucleicacid.

[0126] Another aspect of the present invention is a method of preparinga DNA partial duplex having a portion at one end that has twonon-complementary single stranded sequences. FIG. 7 depicts such amethod in accordance with the present invention. A medium containing asingle stranded polynucleotide SSP is combined with a primer P1 that hasa 3′-end portion Pa, which binds to a sequence that is 8 to 60nucleotides from the 3′-end of SSP. Primer P1 also has an 8 to 60nucleotide portion B1 that does not bind to SSP. The medium is subjectedto conditions under which P1 binds to and is extended along the singlestranded polynucleotide. The complement of SSP is formed but not a copythereof. The conditions employed are as described above for chainextension of a primer only, not as part of an amplification. A duplex B′is formed and contains non-complementary end portions B1 and B2.

[0127] As mentioned above, the present invention also provides fordetection of a target sequence using PCR. An example of this embodimentis depicted in FIG. 8. This PCR method involves formation of afour-strand structure or complex as above for the detection of amutation. However, in the approach in FIG. 8 the target nucleic acidsequence A is the sequence to be detected by PCR and the referencenucleic acid sequence B is introduced as a reagent and contains adifference Q from the target nucleic acid sequence. This difference isas described above for two related nucleic acid sequences. Thus, in thisembodiment the identity of the target nucleic acid sequence is known tothe extent necessary to allow the preparation of the primers and thereference nucleic acid sequence. The formation of such complex involvesproducing two partial duplexes by amplification by using three differentprimers in the polymerase chain reaction and allowing the amplifiedproducts to anneal. In this particular embodiment the formation of thecomplex is dependent on the presence of the target nucleic acidsequence. If the target nucleic acid sequence is not present, no complexis detected. However, when the target nucleic acid is present, there isa difference between the two hybridized portions of the complex. Thecomplex does not dissociate and can be detected as an indication of thepresence of the target nucleic acid sequence.

[0128] Referring now to FIG. 8, target nucleic acid A, if present, isamplified by the polymerase chain reaction using primers P1 and P2 toproduce an amplicon AA. Primer P2 contains a label L1 and primer P1 iscomprised of a 3′-end portion Pa that can hybridize with the targetsequence and 5′-end portion B1 that cannot hybridize with the targetsequence. The amplification is carried out under the reaction conditionsemployed in PCR in the presence of a nucleotide polymerase andnucleoside triphosphates using temperature cycling. Amplicon AA has twostrands, a labeled strand derived from primer P2 and an unlabeled strandderived from primer P1. The unlabeled strand has a 5′-end portion B1 ofprimer P1 and the labeled strand has a corresponding 3′-end portion A2,which is the complement of B1.

[0129] A chain extension of primer P3 along the labeled strand ofamplicon AA is then carried out to produce tailed target partial duplexA′. Primer P3 is comprised of a 3′-end portion Pa, which is identical toPa of primer P1 and which binds to the labeled strand of M. P3 has5′-end portion A1 that is not complementary to amplicon AA. The chainextension is carried out in the presence of a nucleotide polymerase andnucleoside triphosphates under appropriate temperature conditions sothat only the complementary strand of the labeled strand is produced andnot a copy. This complementary unlabeled strand of tailed target partialduplex A′ has a 5′-end portion A1, which is not complementary to the3′-end portion A2 of the labeled strand of A′. Unless the PCR reactionis carried out to produce an excess of the labeled strand, there willalso be present the unlabeled strand from the amplification. This strandis not a template during chain extension to form partial duplex A′.

[0130] In the embodiment of FIG. 8, reference nucleic acid sequence B isin a separate medium, using primer P2 and primer P3, by polymerase chainreaction to produce amplicon BB. The amplification is carried out usingtemperature cycling under the conditions described above in the presenceof a nucleotide polymerase and nucleoside triphosphates. B is comprisedof a sequence identical to A except for difference Q. Generally, primerP2 used for this amplification contains a label L2 that may be the sameas or different than L1. Amplicon BB has two strands, a labeled strandderived from primer P2 and an unlabeled strand derived from primer P3.The unlabeled strand has end portion A1 of primer P3 and the labeledstrand has corresponding end portion B2, which is the complement of A1.

[0131] A chain extension of primer P1 along the labeled strand ofamplicon BB is carried out, under the conditions mentioned above for thechain extension of primer P3 along the labeled strand in duplex AA, toproduce tailed reference partial duplex B′. As mentioned above, primerP1 is comprised of portion Pa, which binds to the labeled strand of BBand portion B1 that does not bind to amplicon BB. The chain extension iscarried out in the presence of a nucleotide polymerase and nucleosidetriphosphates under appropriate temperature conditions so that only thecomplement of the labeled strand is produced and not a copy. Theextended primer P1 has a 5′-end portion B1, which is not complementaryto end portion B2 of the labeled strand of B′. As can be seen, A′ and B′are related in that each of their labeled strands is complementary tothe unlabeled strand of the other except for difference Q.

[0132] The strands of partial duplexes A′ and B′ are allowed to bind andundergo branch migration by combining the mixtures containing partialduplexes A′ and B′ and incubating the combination under conditionsdescribed above for mutation detection wherein complex C′ is formed ifthe target nucleic acid sequence is present. Oligonucleotide tail A1 ofA′ is hybridized to corresponding oligonucleotide tail B2 of B′ and,similarly, oligonucleotide tail A2 of A′ is hybridized tooligonucleotide tail B1 of B′. Branch migration within complex C′continues until difference Q is reached, at which point migrationceases. In the embodiment depicted in FIG. 8, labels L1 and L2 areincorporated into the partial duplexes that comprise complex C′.

[0133] In the embodiment of FIG. 8, the reactions are carried outindependently to produce tailed partial duplexes A′ and B′,respectively. Then, the reaction mixtures can be combined to allow therespective strands of A′ and B′ to bind to one another to form complexC′.

[0134] It is a particularly attractive feature of the present inventionthat the method for the use of PCR in the detection of a target nucleicacid sequence can be carried out in a single reaction container withouta separation step. In this embodiment, a combination is provided in asingle medium. The combination comprises (i) a sample suspected ofcontaining a target nucleic acid sequence, (ii) a reference nucleic acidsequence, related to but different from the target nucleic acidsequence, (iii) a nucleotide polymerase, (iv) nucleoside triphosphates,and (v) primers P1, P2 and P3, wherein P2 may include primer P2 labeledwith L1 and primer P2 labeled with L2, or P2 may be unlabeled andprimers P1 and P3 may be labeled respectively with L1 and L2. The mediumis then subjected to multiple temperature cycles of heating and coolingto simultaneously achieve all of the amplification and chain extensionreactions described above for FIG. 8 except that in this embodimentthere is no need to avoid making copies of any of the extended primers.The medium is subjected to conditions for conducting PCR as describedabove.

[0135] When target nucleic acid is present, all possible partial andcomplete duplexes are formed that can form from 1) single strands thathave any combination of reference or target sequences and 5′-ends A2 andB2, and 2) single strands having any combination of reference or mutantsequences and 5′-ends A1 or B1 wherein the strands may further belabeled with either L1 or L2 when L1 and L2 are different. Among thepartial duplexes that are formed are the tailed partial duplexes A′ andB′, which can bind to each other to form complex C′, which does notdissociate. A determination of the presence of such a complex is thenmade to establish the presence of the target nucleic acid sequence. Whenprimers P1 and P3 are labeled instead of primer P2, the labels L1 and L2in partial duplexes A′ and B′ are attached to tails A1 and B1,respectively, which still provides for detection of complex C′ whentarget nucleic acid sequence is present. .When target nucleic acidsequence is not present (see FIG. 8A), two duplexes form by virtue ofthe amplification of the reference nucleic acid sequence wherein one canachieve an initial PCR amplification with both sets of primers, namely,P2 and P3 on the one hand (represented by duplex BB in FIG. 8A) and P2and P1 on the other (represented by duplex bb in FIG. 8A). Chainextension of primer P1 on amplicon BB produces B′, and chain extensionprimer P3 on amplicon bb produces b′. Any four strand structure formedby hybridization of the respective tails of B′ and b′ to one anothercompletely dissociates because there is no difference in either of theduplexes to inhibit complete strand exchange. In other words, thecomplex dissociates into normal duplex structures D′ and E′ by strandexchange by means of branch migration when the hybridized portions ofeach partial duplex are identical. In this embodiment in the absence oftarget nucleic acid sequence, the hybridized portions are identical inthat each strand contains difference Q.

[0136] As a matter of convenience, predetermined amounts of reagentsemployed in the present invention can be provided in a kit in packagedcombination. A kit can comprise in packaged combination (a) a primer P2that is extendable along one of the strands of the target and referencenucleic acid sequences, (b) a primer P1 comprising a 3′-end portion Pathat binds to and is extendable along the other of the strands of thetarget and reference nucleic acid sequences and a 5′-end portion B1 thatdoes not bind to the target and reference nucleic acid sequences, and(c) a primer P3 comprising 3′-end portion Pa and a portion A1 that isdifferent from B1 and does not bind to the target and reference nucleicacid sequences. Preferably, primer P2 can be labeled, but primers P1 andP3 alternatively may be labeled. The kit can also include a referencenucleic acid, which corresponds to a target nucleic acid sequence exceptfor the possible presence of a difference such as a mutation, andreagents for conducting an amplification of target nucleic acid sequenceprior to subjecting the target nucleic acid sequence to the methods ofthe present invention. The kit can also include nucleoside triphosphatesand a nucleotide polymerase. The kit can further include two additionaloligonucleotide primers PX1 and PX2 where the primers are related inthat a product of the extension of one along the target sequence servesas a template for the extension of the other. The kit can furtherinclude particles as described above capable of binding to the label onat least one of the primers. The kit can further include members of asignal producing system and also various buffered media, some of whichmay contain one or more of the above reagents. Preferably, primers PX1,PX2, P1, P2 and P3 are packaged in a single container. More preferably,at least all of the above components other than buffer are packaged in asingle container.

[0137] The kit can further include a pair of adapter primers foramplifying the target and reference nucleic acids. One of the primershas a 3′-end portion that is hybridizable to the target and referencenucleic acids and a portion 5′ thereof that is not hybridizable with thetarget or reference nucleic acids and is substantially identical toprimer P2. The other of the primers has a 3′-end portion that ishybridizable to the target and reference nucleic acids and a portion 5′thereof that is not hybridizable with the target or reference nucleicacids and is substantially identical to the 3′-end portion Pa of primersP1 and P3. The adapter primers are usually packaged in a containerseparate from primers P1, P2 and P3.

[0138] The relative amounts of the various reagents in the kits can bevaried widely to provide for concentrations of the reagents whichsubstantially optimize the reactions that need to occur during thepresent method and to further substantially optimize the sensitivity ofthe method in detecting a mutation. Under appropriate circumstances oneor more of the reagents in the kit can be provided as a dry powder,usually lyophilized, including excipients, which on dissolution willprovide for a reagent solution having the appropriate concentrations forperforming a method or assay in accordance with the present invention.Each reagent can be packaged in separate containers or some or all ofthe reagents can be combined in one container where cross-reactivity andshelf life permit. In a particular embodiment of a kit in accordancewith the present invention, the reagents are packaged in a singlecontainer. The kits may also include a written description of a methodin accordance with the present invention as described above.

EXAMPLES

[0139] The invention is demonstrated further by the followingillustrative examples. Temperatures are in degrees centigrade (OC) andparts and percentages are by weight, unless otherwise indicated. Thefollowing definitions and abbreviations are used herein:

[0140] Tris—Tris(hydroxymethyl)aminomethane-HCl (a 10×solution) fromBioWhittaker, Walkersville, Md.

[0141] BSA—bovine serum albumin from Gibco BRL, Gaithersburg Md.

[0142] bp—base pairs

[0143] wt (+)—wild type allele

[0144] mut (−)—mutant allele

[0145] +/+—homozygote with 2 normal alleles

[0146] −/−—homozygote with 2 mutant alleles

[0147] +/−—heterozygote with 1 normal and 1 mutant allele

[0148] Target sample—DNA sample to be tested for the presence of amutation;

[0149] Reference sample—DNA sample homozygous for the wt sequence withwhich target samples are challenged.

[0150] sec—seconds

[0151] hr—hours

[0152] min—minutes

[0153] Buffer A—10 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM MgCl₂, 200Wg/ml BSA

[0154] Buffer B—10 mM Tris-HCl (pH 8.3), 50 mM KCl, 20 mM MgCl₂, 200tg/ml BSA

[0155] Buffer C—0.1M Tris, 0.3M NaCl, 25 mM EDTA, 0.1% BSA, 0.1% dextranT-500, a 1:320 dilution of mouse IgG (HBR-1 from Scantibodies LaboratoryInc., Los Angeles, Calif.), 0.05% Kathon (Rohm and Haas, Philadelphia,Pa.), and 0.01% gentamycin sulfate.

[0156] RLU—relative light units

[0157] nt—nucleotides

[0158] MAD—maleimidylaminodextran

[0159] Ab—antibody

[0160] Sav—streptavidin

[0161] MOPS—3-(N-morpholino)propane sulfonic acid

[0162] hr—hour

[0163] sulfo-SMCC—sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate

[0164] NHS—N-hydroxysuccinimide

[0165] EDAC—1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0166] DMSO—dimethylsulfoxide

[0167] MES—morpholinoethanesulfonate

[0168] rpm—rotations per min

[0169] EDTA—ethylenediaminetetraacetic acid

[0170] SATA—N-succinimidyl S-acetylthioacetate

[0171] BSA—bovine serum albumin from Sigma Chemical Company, St. LouisMo.

[0172] eq—equivalents

[0173] bp—base pairs

[0174] A₂₈₀—absorbance at wavelength 280 nanometers

[0175] DexAl—dextran aldehyde

[0176] DPP—4,7-diphenylphenanthroline

[0177] Eu(TTA)₃—europium tri-3-(2-thienoyl)-1,1,1-trifluoroacetonate

[0178] L or l—liter

[0179] exo VII—exonuclease VII from E.coli (from Amersham Life Science)(USB).

[0180] DMF—dimethyl formamide

[0181] THF—tetrahydrofuran

[0182] MS—mass spectroscopy

[0183] NMR—nuclear magnetic resonance spectroscopy

[0184] TMSCl—tetramethylsilylchloride

[0185] ELISA—enzyme linked immunosorbent assay as described in“Enzyme-Immunoassay,” Edward T. Maggio, CRC Press, Inc., Boca Raton,Fla. (1980)

[0186] Monoclonal antibodies were produced by standard hybrid celltechnology. Briefly, the appropriated immunogen was injected into ahost, usually a mouse or other suitable animal, and after a suitableperiod of time the spleen cells from the host were obtained.Alternatively, unsensitized cells from the host were isolated anddirectly sensitized with the immunogen in vitro. Hybrid cells wereformed by fusing the above cells with an appropriate myeloma cell lineand culturing the fused cells. The antibodies produced by the culturedhybrid cells were screened for their binding affinity to the particularantigen, dig-BSA conjugate. A number of screening techniques wereemployed such as, for example, ELISA screens. Selected fusions were thenrecloned.

[0187] Beads:

[0188] Acc-Ab_(Dig)—Acceptor beads coupled (MAD) to the anti-Digantibody (with 377 antibody molecules per bead) were prepared asfollows:

[0189] Hydroxypropylaminodextran (1 NH₂/7 glucose) was prepared bydissolving Dextran T-500 (Pharmacia, Uppsala, Sweden) (50 g) in 150 mLof H₂O in a 3-neck round-bottom flask equipped with mechanical stirrerand dropping funnel. To the above solution was added 18.8 g of Zn (BF₄)₂and the temperature was brought to 87° C. with a hot water bath.Epichlorohydrin (350 mL) was added dropwise with stirring over about 30min while the temperature was maintained at 87-88° C. The mixture wasstirred for 4 hr while the temperature was maintained between 80° C. and950C, then the mixture was cooled to room temperature. Chlorodextranproduct was precipitated by pouring slowly into 3 L of methanol withvigorous stirring, recovered by filtration and dried overnight in avacuum oven.

[0190] The chlorodextran product was dissolved in 200 mL of water andadded to 2 L of concentrated aqueous ammonia (36%). This solution wasstirred for 4 days at room temperature, then concentrated to about 190mL on a rotary evaporator. The concentrate was divided into two equalbatches, and each batch was precipitated by pouring slowly into 2 L ofrapidly stirring methanol. The final product was recovered by filtrationand dried under vacuum.

[0191] Hydroxypropylaminodextran (1 NH₂/7 glucose), prepared above, wasdissolved in 50 mM MOPS, pH 7.2, at 12.5 mg/mL. The solution was stirredfor 8 hr at room temperature, stored under refrigeration and centrifugedfor 45 min at 15,000 rpm in a Sorvall RC-5B centrifuge immediatelybefore use to remove a trace of solid material. To 10 mL of thissolution was added 23.1 mg of Sulfo-SMCC in 1 mL of water. This mixturewas incubated for 1 hr at room temperature and used without furtherpurification.

[0192] C-28 thioxene was prepared as follows:

[0193] To a solution of 4-bromoaniline (30 g, 174 mmol) in dry DMF (200mL) was added 1-bromotetradecane (89.3 mL, 366 mmol) andN,N-diisopropylethylamine (62.2 mL, 357 mmol). The reaction solution washeated at 90° C. for 16 hr under argon before being cooled to roomtemperature. To this reaction solution was again added1-bromotetradecane (45 mL, 184 mmol) and N,N-diisopropylethylamine (31mL, 178 mmol) and the reaction mixture was heated at 90° C. for another15 hr. After cooling, the reaction solution was concentrated in vacuoand the residue was diluted with CH₂Cl₂ (400 mL). The CH₂Cl₂ solutionwas washed with 1 N aqueous NaOH (2×), H₂O, and brine, was dried overNa₂SO₄ and was concentrated in vacuo to yield a dark brown oil (about110 g). Preparative column chromatography on silica gel by a Waters 500Prep LC system eluting with hexane afforded a yellow oil that containedmainly the product (4-bromo-N,N-di-(C₁₄H₂₉)-aniline) along with a minorcomponent 1-bromotetradecane. The latter compound was removed from themixture by vacuum distillation (bp 105-110° C., 0.6 mm) to leave 50.2 g(51%) of the product as a brown oil. To a mixture of magnesium turnings(9.60 g, 395 mmol) in dry THF (30 mL) under argon was added dropwise asolution of the above substituted aniline product (44.7 g, 79 mmol) inTHF (250 mL). A few crystals of iodine were added to initiate theformation of the Grignard reagent. When the reaction mixture became warmand began to reflux, the addition rate was regulated to maintain agentle reflux. After addition was complete, the mixture was heated atreflux for an additional hour. The cooled supernatant solution wastransferred via cannula to an addition funnel and added dropwise (over2.5 hr) to a solution of phenylglyoxal (11.7 g, 87 mmol) in THF (300 mL)at −30° C. under argon. The reaction mixture was gradually warmed to 0°C. over 1 hr an stirred for another 30 min. The resulting mixture waspoured into a mixture of ice water (800 mL) and ethyl acetate (250 mL).The organic phase was separated and the aqueous phase was extracted withethyl acetate (3×). The combined organic phases were washed with H₂O(2×), brine and was dried over MgSO₄. Evaporation of the solvent gave48.8 g of the crude product as a dark green oily liquid. Flash columnchromatography of this liquid (gradient elution with hexane, 1.5:98.5,3:97, 5:95 ethyl acetate:hexane) afforded 24.7 g (50%) of the benzoinproduct (MS (C₄₂H₆₉NO₂): [M-H]⁺ 618.6, ¹H NMR (250 MHz, CDCl₃) wasconsistent with the expected benzoin product. To a solution of thebenzoin product from above (24.7 g, 40 mmol) in dry toluene (500 mL) wasadded sequentially 2-mercaptoethanol (25 g, 320 mmol) and TMSCl (100 mL,788 mmol). The reaction solution was heated at reflux for 23 hr underargon before being cooled to room temperature. To this was addedadditional TMSCl (50 mL, 394 mmol);and the reaction solution was heatedat reflux for another 3 hr. The resulting solution was cooled, was madebasic with cold 2.5N aqueous NaOH and was extracted with CH₂Cl₂ (3×).The combined organic layers were washed with saturated aqueous NaHCO₃(2×) and brine, was dried over Na₂SO₄ and was concentrated in vacuo togive a brown oily liquid. Preparative column chromatography on silicagel by using a Waters 500 Prep LC system (gradient elution with hexane,1:99, 2:98 ethyl acetate:hexane) provided 15.5 g (60%) of the C-28thioxene as an orange-yellow oil (MS (C₄₄H₇₁NOS): [M-H]⁺661.6, ¹H NMR(250 MHz, CDCl₃) was consistent with the expected C-28 thioxene product2-(4-(N,N-di-(C₁₄H₂₉)-anilino)-3-phenyl thioxene.

[0194] Carboxyl acceptor beads were prepared as follows: The startingbeads were carboxylate modified latex purchased from Seradyn ParticleTechnology, Indianapolis, Ind. The beads contained Eu(TTA)₃DPP preparedas follows: DPP/Eu(TTA)₃ was prepared by combining 8.69 g of Eu(TTA)₃.3H₂O (10 mmoles, Kodak Chemical Company, Rochester NY) and 1.8 g of1,10-phenanthroline (10 mmoles, Aldrich) in 50 ml of dry toluene andheating to 95° C. in an oil bath for one 1 hour. Toluene was removedunder reduced pressure. The ash colored solid was crystallized from 10ml of toluene to yield 10 grams of DPP/Eu(TTA)₃. Absorption spectrum:270 nm (20,000), 340 nm (60,000) (Toluene) 1.R(KBr): Cm⁻¹: 3440(s),1600(s), 1540(s), 1400(s), 1300(s). Four mL of 20% suspension (400 mg)of washed 175 nm carboxylate modified latex was diluted with 3 mL ofethoxyethanol in a 25 mL round bottom (R.B.) flask with a stir bar. TheR.B. flask was then placed in an oil bath at 1050C and stirred for 10minutes. Then, 3.3 mM C-28 thioxene and 15.5 mM Eu(TTA)₃DPP was added;the beads were stirred for 5 minutes more. At this point 1.0 mL of 0.1 NNaOH was added slowly over 5 minutes. During all the additions, the oilbath temperature was maintained at 105° C. The oil bath temperature wasslowly allowed to drop to room temperature over 2 hours. After cooling,the mixture was diluted with 20 mL of ethanol and centrifuged (12,500rpm, 30 minutes). Supernatants were discarded and the pelletsresuspended in ethanol by sonication. Centrifugation was repeated, andthe pellet was resuspended in water; and centrifugation was repeated.The pellet was resuspended in 5 mL of aqueous ethanol to a final volumeof 40 mL.

[0195] Carboxyl acceptor beads prepared above (99 mg in 4.5 mL water)were added slowly with vortexing to 5.5 mL of MAD aminodextran fromabove, followed by 1 mL of 200 mg/mL NHS in 50 mM MES, pH 6,1 mL of 200mg/mL EDAC in water, and 450 μL of 1 M HCl, final pH 6. The mixture wasincubated overnight at room temperature in the dark, then reacted with200 mg succinic anhydride in 0.5 mL of DMSO for 30 min at roomtemperature. Freshly opened Surfact-Amps Tween-20 (Pierce ChemicalCompany, Rockford, Ill.) was added and the beads were centrifuged 30 minat 15,000 rpm in a Sorvall RC-5B centrifuge, washed by centrifugationwith three 10 mL portions of 50 mM MOPS, 50 mM EDTA, 0.1% Surfact-AmpsTween-20 (Pierce Chemical Company), pH 7.2, and resuspended in 3 mL ofthe same.

[0196] Monoclonal anti-digoxin Ab (prepared as described above) waspurified by ABx resin (Baker Chemical Company, Phillipsburg, N.J.) andwas dialyzed into 0.15 M NaCl, 5 mM Na₂HPO₄, pH 7.4. The anti-digoxin Abwas thiolated by mixing 622 μL (4.28 mg) with 10.2 AL of SATA (1.25mg/mL in ethanol, 2 eq.), incubating for 1 hr at room temperature anddialyzing cold against 2×2 L of 150 mM NaCl, 10 mM Na₂HPO₄, 1 mM EDTA,pH7. The thioacetylated antibody was deacetylated by adding 62.2 μL ofhydroxylamine (1 M H₂NOH, 50 mM MOPS, 25 mM EDTA, pH 7), bubbling withargon and incubating for 1 hr at room temperature. The product wasapplied to a Pharmacia PD-10 column (G-25) and eluted with 50 mM MOPS,50 mM EDTA, pH 7.2, bubbled with argon. After 2.5 mL fore-run, three-1mL fractions were collected and combined. Recovery of antibody was 3.66mg or 86% by A₂₈₀. Surfact-Amps Tween-20 (10%) was added to give 0.2%final concentration.

[0197] A 1.4 mL aliquot of the thiolated antibody above (1.71 mgantibody) was immediately added to 300 μL (10 mg) of maleimidated beadsprepared above plus enough 10% Tween-20 to bring final concentration ofthe mixture to 0.2%. The tube was purged with argon and incubatedovernight at room temperature in the dark. To the above was added 3.4 μLof 1 M HSCH₂COOH in water. After 30 min at room temperature, 6.8 μL ofICH₂COOH (1 M in water) was added. After 30 min 3.5 mL of 0.17M glycine,0.1 M NaCl, 0.1% (v/v) Tween-20, 10 mg/mL BSA, pH 9.2 was added and thebeads were centrifuged (30 min at 15,000 rpm), incubated for 3hr in 5 mLof the same buffer, centrifuged, washed by centrifugation with three-5mL portions of Buffer C, resuspended in 5 mL of Buffer C and storedunder refrigeration. The size of the beads, determined in Buffer C, was301+/−56 nm. Binding capacity was determined with 1251-digoxin and wasequivalent to 377 antibody molecules per bead.

[0198] Silicon tetra-t-butyl phthalocyanine was prepared as follows:Sodium metal, freshly cut (5.0 g, 208 mmol), was added to 300 mL ofanhydrous ether in a two-liter, 3-necked flask equipped with a magneticstirrer, reflux condenser, a drying tube and a gas bubbler. After thesodium was completely dissolved, 4-t-butyl-1,2-dicyanobenzene (38.64g,210 mmol, from TCI Chemicals, Portland OR) was added using a funnel. Themixture became clear and the temperature increased to about 50° C. Atthis point a continuous stream of anhydrous ammonia gas was introducedthrough the glass bubbler into the reaction mixture for 1 hr. Thereaction mixture was then heated under reflux for 4 hr. while the streamof ammonia gas continued. During the course of the reaction, as solidstarted to precipitate. The resulting suspension was evaporated todryness (house vacuum) and the residue was suspended in water (400 mL)and filtered. The solid was dried (60° C., house vacuum, P₂O₅). Theyield of the product (1,3-diiminoisoindoline, 42.2 g) was almostquantitative. This material was used for the next step without furtherpurification. To a one-liter, three-necked flask equipped with acondenser and a drying tube was added the above product (18 g, 89 mmol)and quinoline (200 mL, Aldrich Chemical Company, St. Louis Mo.). Silicontetrachloride (11 mL, 95 mmol, Aldrich Chemical Company) was added witha syringe to the stirred solution over a period of 10 minutes. After theaddition was completed, the reaction mixture was heated to 180-185° C.in an oil bath for 1 hr. The reaction was allowed to cool to roomtemperature and concentrated HCl was carefully added to acidify thereaction mixture (pH 5-6). The dark brown reaction mixture was cooledand filtered. The solid was washed with 100 mL of water and dried (housevacuum, 60° C., P₂O₅). The solid material was placed in a 1-liter, roundbottom flask an concentrated sulfuric acid (500 mL) was added withstirring. The mixture was stirred for 4 hr. at 60° C. and was thencarefully diluted with crushed ice (2000 g). The resulting mixture wasfiltered and the solid wad washed with 100 mL of water and dried. Thedark blue solid was transferred to a 1-liter, round bottom flask,concentrated ammonia (500 mL) was added, and the mixture was heated andstirred under reflux for 2 hr., was cooled to room temperature and wasfiltered. The solid was washed with 50 mL of water and dried undervacuum (house vacuum, 60° C., P₂O₅) to give 12 g of product silicontetra-t-butyl phthalocyanine as a dark blue solid. 3-picoline (12 g,from Aldrich Chemical Company), tri-n-butyl amine (anhydrous, 40 mL) andtri-n-hexyl chlorosilane (11.5 g) were added to 12 g of the aboveproduct in a one-liter, three-necked flask, equipped with a magneticstirrer an a reflux condenser. The mixture was heated under reflux for1.5 hr. an then cooled to room temperature. The picoline was distilledoff under high vacuum (oil pump at about 1 mm Hg) to dryness. Theresidue was dissolved in CH₂Cl₂ and purified using a silica gel column(hexane) to give 10 g of pure product di-(tri-n-hexylsilyl)-silicontetra-t-butyl phthalocyanine as a dark blue solid. (MS: [M-H]⁺1364.2,absorption spectra: methanol: 674 nm (ε 180,000): toluene 678 nm, ¹H NMR(250 MHz, CDCl₃): δ: −2.4(m,12H), −1.3(m, 12H), 0.2-0.9 (m, 54H), 1.8(s,36H), 8.3(d, 4H) and 9.6 (m, 8H) was consistent with the above expectedproduct.

[0199] Sens-Sav—Sensitizer beads coupled to Streptavidin (2300Sav/bead). The sensitizer beads were prepared placing 600 mL ofcarboxylate modified beads (Seradyn) in a three-necked, round-bottomflask equipped with a mechanical stirrer, a glass stopper with athermometer attached to it in one neck, and a funnel in the oppositeneck. The flask had been immersed in an oil bath maintained at 94+/−1°C. The beads were added to the flask through the funnel in the neck andthe bead container was rinsed with 830 mL of ethoxyethanol, 1700 mL ofethylene glycol and 60 mL of 0.1N NaOH and the rinse was added to theflask through the funnel. The funnel was replaced with a 24-40 rubberseptum. The beads were stirred at 765 rpm at a temperature of 94+/−1° C.for 40 min.

[0200] Silicon tetra-t-butyl phthalocyanine (10.0 g) was dissolved in300 mL of benzyl alcohol at 60+/−5° C. and 85 mL was added to the aboveround bottom flask through the septum by means of a syringe heated to120+/−10° C. at a rate of 3 mL per min. The remaining 85 mL of thephthalocyanine solution was then added as described above. The syringeand flask originally containing the phthalocyanine was rinsed with 40 mLof benzyl alcohol and transferred to round-bottom flask. After 15 min900 mL of deionized water and 75 mL of 0.1N NaOH was added dropwise over40 min. The temperature of the oil bath was allowed to drop slowly to40+/−10° C. and stirring was then discontinued. The beads were thenfiltered through a 43 micron polyester filter and subjected to aMicrogon tangential flow filtration apparatus (Microgon Inc., LagunaHills, Calif.) using ethanol:water, 100:0 to 10:90, and then filteredthrough a 43 micron polyester filter.

[0201] Sulfo-SMCC (11.55 mg) was dissolved in 0.5 mL distilled water.Slowly, during 10 sec, the above solution was added to 5 mL of stirringaminodextran (Molecular Probes, Eugene, Oreg.) solution (12.5 mg/mL in50 mM MOPS, pH 7.2). The mixture was incubated for 1 hr at roomtemperature.

[0202] To the stirring solution above was added 5 mL of 20 mg/mL (100mg) of the sensitizer beads prepared above in distilled water. Then, 1mL of 200 mg/mL NHS (prepared fresh in 50 mM MES, pH adjusted to 6.0with 6N NaOH). 200 mg EDAC was dissolved in 1 mL distilled water andthis solution was added slowly with stirring to the sensitizer beads.The pH was adjusted to 6.0 by addition of 450 μL of 1N HCl and themixture was incubated overnight in the dark. A solution of 100 mg ofsuccinic anhydride in 0.5 mL of DMSO was added to the sensitizer beadsand the mixture was incubated for 30 min at room temperature in thedark. To this mixture was added 0.13 mL 10% Tween-20 bringing the finalconcentration of Tween-20 to 0.1%. The beads were centrifuged for 45 minat 15,000 rpm as above. The supernatant was discarded and the beads wereresuspended in 10 mL of buffer (50 mM MOPS, 50 mM EDTA and 0.1%Tween-20, pH 7.2). The mixture was sonicated to disperse the beads. Thebeads were centrifuged for 30 min as described above, the supernatantwas discarded and the beads were resuspended. This procedure wasrepeated for a total of three times. Then, the beads were resuspended to40 mg/mL in 2.5 mL of the above buffer, saturated with argon andTween-20 was added to a concentration of 0.1%. The beads were stored at4° C.

[0203] Streptavidin was bound to the above beads using 25 mgstreptavidin for 100 mg of beads. 25 mg streptavidin (50 mg Aaston solidfrom Aaston, Wellesley, Mass.) was dissolved in 1 mL of 1 mM EDTA, pH7.5, and 77 μL of 2.5 mg/mL SATA in ethanol was added thereto. Themixture was incubated for 30 min at room temperature. A deacetylationsolution was prepared containing 1M hydroxylamine-HCl, 50 mM Na₂PO₄, 25mM EDTA, pH 7.0. 0.1 mL of this deacetylation solution was added to theabove solution and incubated for 1 hr at room temperature. The resultingthiolated streptavidin was purified on a Pharmacia PD10 column andwashed with a column buffer containing 50 mM MOPS, 50 mM EDTA, pH 7.2.The volume of the sample was brought to 2.5 mL by adding 1.5 mL of theabove column buffer. The sample was loaded on the column and eluted with3.5 mL of the column buffer. The thiolated streptavidin was diluted to 5mL by adding 1.5 mL of 50 mM MOPS, 50 mM EDTA, 0.1% Tween-20, pH 7.2. 5mL of the thiolated streptavidin solution was added to 5 mL of thesensitizer beads, under argon, and mixed well. The beads were toppedwith argon for 1 min, the tube was sealed and the reaction mixture wasincubated overnight at room temperature in the dark.

[0204] To the above beads was added 7.5 mL of 50 mM MOPS, 50 mM EDTA,0.1% Tween-20, pH 7.2 to bring the beads to 1 mg/mL. The remainingmaleimides were capped by adding mercaptoacetic acid at a finalconcentration of 2 mM. The mixture was incubated in the dark for 30 minat room temperature. The remaining thiols were capped by addingiodoacetic acid at a final concentration of 10 mM and the mixture wasincubated at room temperature for 30 min in the dark. The beads werecentrifuged for 30 min at 15,000 rpm as above for a total of threetimes.

Example 1 Detection of Difference in Nucleic Acid Using Step-wiseApproach

[0205] Genomic DNA having the following point mutations within exon 11of the CFTR gene used herein:

[0206] Heterozygous DNA with one wild type (wt) allele and one of thefollowing mutant alleles:

[0207] G542X (G>T substitution) from Roche Molecular Systems, Alameda,Calif.;

[0208] G551 D (G>A substitution) from Roche Molecular Systems, Alameda,Calif.;

[0209] R553X (C>T substitution) from Roche Molecular Systems, Alameda,Calif.;

[0210] R560T (G>C substitution) from Roche Molecular Systems, Alameda,Calif.

[0211] Homozygous DNA:

[0212] G542X/G542X from Coriell Institute for Medical Research, Camden,N.J.

[0213] Primers:

[0214] PX2—forward PCR primer outside the sequence to be tested; 5°C.AACTGTGGTTAAAGCAATAGTGTGATATATGA 3′ (SEQ ID NO: 1) from Oligos Etc.,Inc., Wilsonville, Oreg.

[0215] PX1—reverse PCR primer outside the sequence to be tested;5′GCACAGATTCTGAGTAACCATAATCTCTACCA3′ (SEQ ID NO: 2) from Oligos Etc.,Inc., Wilsonville, Oreg.

[0216] P2—forward PCR primer; 5′GCCTTTCAAATTCAGATTGAGC 3′ (SEQ ID NO: 3)from Oligos Etc., Inc., Wilsonville, Oreg.

[0217] P2B—P2 biotinylated at the 5′-end from Genosys Biotechnologies,Inc., The Woodlands, Tex.;

[0218] P2D—P2 Dig-labeled at the 5′-end from Genosys Biotechnologies,Inc., The Woodlands, Tex.;

[0219] Pa—reverse PCR primer; 5′GACATTTACAGCAAATGCTTGC3′ (SEQ ID NO: 4)from Oligos Etc., Inc., Wilsonville, Oreg.

[0220] P1—reverse PCR primer which has a 3′ end that is identical withPa, but which has a different 5′-“tail” B1 (20 nucleotides long). The“tails” are arbitrary sequences that are not complementary to thegenomic sequence; 5′ACCATGCTCGAGATTACGAGGACATTTACAGCAAATGCTTGC3′ (SEQ IDNO: 5) from Genosys Biotechnologies, Inc., The Woodlands, Tex.

[0221] P3—reverse PCR primer which has a 3′ end that is identical withPa but which has a different 5′-“tail” A1 (20 nucleotides long). The“tails” are arbitrary sequences that are not complementary to thegenomic sequence; 5′GATCCTAGGCCTCACGTATTGACATTTACAGCAAATGCTTGC3′ (SEQ IDNO: 6) from Genosys Biotechnologies, Inc., The Woodlands, Tex.

[0222] Step 1 (1st-round PCR).

[0223] Two samples containing 10-100 ng genomic DNA from humanperipheral blood, one a target sample and the other a reference sample(wild type sample) were amplified using the P2/Pa pair of primersencompassing the hot spot mutation site in exon 11 of the CFTR (cysticfibrosis) gene. A total of 30 cycles in the Ericomp® thermocycler (fromEricomp, San Diego, Calif.) were performed consisting of a 30-secdenaturation step at 94° C., a 1-min reannealing step at 64° C. and a1-min extension step at 72° C., preceded by a 2-min denaturation ofgenomic DNA at 95° C. and followed by a 4-min final extension at 72° C.The reaction volume was 100 Fl and the buffer was Buffer A. Theconcentration of each primer was 250 nM (as below, in steps 2 and 4). Aproof-reading thermostable polymerase (Pfu® DNA polymerase, StratageneCloning Systems, La Jolla, Calif.) was used. A hot-start technique usingTM HotStart 100™ reaction tubes (Molecular Bio-Products, Inc., SanDiego, Calif.) was utilized. The resulting PCR product was 203 bp inlength.

[0224] Step 2 (2nd-round PCR).

[0225] The reaction mixtures from above were diluted 1:1000, and 2 μl ofthese dilutions were used in 100 μl reactions with the primers P2B andP1 for the target sample and P2D and P3 for the reference sample. 20amplification cycles were run under the same conditions as in Step 1.

[0226] Step 3 (Removal of Primers).

[0227] 0.5 μl (5 U) of exo VII was added to each reaction mixture. ExoVII is a strictly single-strand specific enzyme, with both 5′ to 3′ and3′ to 5′ exonuclease activities. The digestion of the free primers wascarried out for 30 min at 37° C., followed by the inactivation of theenzyme (10 min at 95° C.), as described by Li, et al., (1991) NucleicAcids Research 19, 3139-3141: Eliminating Primers from CompletedPolymerase Chain Reactions with Exonuclease VII.

[0228] Step 4 (Addition of Tails).

[0229] Primers P3 (for the test sample) and P1 (for the referencesample) were added to the reaction mixtures, and the mixtures weresubjected to one PCR cycle (30 sec at 94° C., 1 min at 64° C. and 1 minat 72° C.). Usually fresh Pfu DNA polymerase was also added, but thiswas not obligatory. Inclusion of primer P2 in this step is optional.

[0230] Step 5 (Branch Migration).

[0231] 2 μl of each of the reaction mixtures was combined and 8 μl ofbuffer A containing 28 mM MgCl₂ was added (final concentration 20 mMMgCl₂). The reaction mixture was overlaid with 5 μl mineral oil andincubated 30 min at 65° C. Other MgCl₂ concentrations (4 mM and 10 mM)were also tested and found to support branch migration. The reaction wasalso repeated at other incubation temperatures (50, 60 and 70° C.).

[0232] Step 6 (Detection).

[0233] Acc-Ab_(Dig) and Sens-Sav beads were titrated with varyingamounts of the branch migration reaction mixtures with varying ratios oftest sample to reference sample to assure a linear response. Amounts ofthe components were as follows.

[0234] A 2 μl aliquot of the branch migration reaction mixture wascombined with 100 μl Buffer B containing 5 μl (10 μg) Sens-Sav and 5 μl(10 gg) Acc-Ab_(Dig) beads and incubated for 5 min at 37° C. Thereaction mixture was then irradiated with a 150 watt Xenon lamp for 3sec (3 cycles of 1 sec illumination and 1 sec waiting time) and thesignal was then read.

[0235] The results of a typical experiment conducted according to theprotocol of this Example 1 were as follows (Table 1): TABLE 1 SampleSignal (RLU) Blank  5368 WT homozygote (+/+)  14556 G542X/G542Xhomozygote (−/−) 739004 G542X/WT heterozygote (+/−) 343206

Example 2 Detection of Difference in Nucleic Acid Using SimplifiedStep-wise Approach

[0236] Step 1 (1st-round PCR)

[0237] Carried out in the same manner as that for Step 1 of Example 1.

[0238] Step 2 (2nd-round PCR).

[0239] The reaction mixtures from Step 1 were diluted 1:1000, and 2 μlof these dilutions were added to 100 μl reaction mixtures containingprimers P2B, P1 and P3 for the test sample and P2D, P1 and P3 for thereference sample. The concentrations of P1 and P3 were 125 nM each. 20amplification cycles were run under the same conditions as in Step 1.

[0240] Step 3 (Branch Migration).

[0241] Note that in contrast to Example 1, removal of the primers wasnot carried out.

[0242] The reaction mixtures were combined as in Step 5 of Example 1.The reaction mixture was heated for 1 min. at 95° C. (denaturation)followed by 30 min. at 65° C. During this incubation period the DNAstrands were allowed to reanneal in all possible combinations. 50% ofdouble-stranded molecules resulting from reannealing were full lengthduplexes and another 50% of the double stranded molecules were partialtailed duplexes. Also during the above incubation, these lattermolecules interacted with each other forming 4-stranded complexes andundergoing branch migration. Among them, only 2 out of 16 possiblecombinations (formed by 1 wild type and I mutant homoduplex labeled withbiotin and digoxin, respectively) were informative and generated signal.The remaining 14 combinations were formed by either 2 homoduplexes ofthe same kind (both wild type or both mutant) or 2 heteroduplexes. Noneof these combinations generated signal because they all undergo completestrand exchange.

[0243] Step 4 (Detection)

[0244] Carried out in the same manner as that for Step 6 of Example 1.

[0245] The results of a typical experiment conducted according toExample 2 were as follows (Table 2): TABLE 2 Sample Signal (RLU) blank 7706 WT homozygote (+/+)  15748 G542X/G542X homozygote (−/−) 730744G542X/WT heterozygote (+/−) 233728

[0246] A panel of 10 samples was screened for mutations in exon 11 ofthe cystic fibrosis gene according to the protocol of this Example 2.All 5 mutants (1 homozygote and 4 heterozygotes) were correctlydetected, as shown in the following table (Table 3). Sample WT1 was usedas the reference sample. TABLE 3 Sample Signal (RLU) blank  6016 WT1(+/+)  17286 WT2 (+/+)  17824 WT3 (+/+)  19318 WT4 (+/+)  18008 WT5(+/+)  17446 G542X/G542X homozygote (−/−) 840476 G542X/WT heterozygote(+/−) 297120 G551D/WT heterozygote (+/−) 379426 R553X/WT heterozygote(+/−) 490572 R560T/WT heterozygote (+/−) 342778

[0247] The differences in signals obtained for the different mutationsreflect slight variations in the amounts of amplicons and not any biasof the method towards particular mutations (in another similarexperiment the signals corresponding to these four heterozygotes werealso slightly different from each other, but the order was not thesame).

Example 3 Detection of Difference in Nucleic Acid Using PartialStep-wise Approach

[0248] The protocol followed in Example 2 was simplified further. Allreactions, with the exception of the detection step, were carried out ina single reaction container. Steps 1 and 2 were combined as step 2 inthis Example 3 so that genomic DNA was used directly as target for thegeneration of the tailed labeled duplexes. In order to reducenon-specific priming of primers P2B, P2D, P1 and P3, a preliminaryamplification of the genomic target was conducted prior to step 2 byalso including primers PX2 and PX1. These primers had higher meltingtemperatures (Tm) than P2B and P2D and P1 and P3, respectively, and eachbound upstream of the respective P2B and P2D and P1 and P3 bindingsites. This procedure is well known (Erlich, et al., Science (1991)252:1643-1651) and the primers that become active only in step 2 beloware known as “nested” or “inner” primers.

[0249] The preliminary amplification was carried out by using anannealing temperature that permitted PX1 and PX2 to bind but was abovethe melting temperatures of the other primers. In this procedure thisinitial PCR amplification was carried out with thermal cycling (25cycles) at 94° and 70° C. for periods of 30 sec and 2 min, respectively.The amplification occurring in steps 1 and 2 above (Examples 1 and 2)with primers P2B (test samples), P2D (reference samples), P1 and P3 wascarried out by continuing thermal cycling with an annealing temperaturethat permitted these primers to bind. This second PCR, step 2, wascarried out with thermal cycling (12 cycles) at 94°, 64°, and 72° C.,for periods of 30 sec, 1 min and 1 min, respectively. The test and thereference samples were mixed and processed as in step 3 of Example 2(denaturation at 94° C. for 1 min and annealing at 65° C. for 30minutes). The association of biotin and digoxin was detected by usingthe signal producing system as in Example 1.

[0250] The results are summarized as follows (Table 4): TABLE 4 SampleSignal (RLU) blank  4846 WT homozygote (+/+)  12058 G542X/G542Xhomozygote (−/−) 200808 G542X/WT heterozygote (+/−)  99752

Example 4 Detection of Difference in Nucleic Acid Using SimultaneousApproach

[0251] The protocol followed in Example 3 was further simplified bycombining the two amplification reactions and carrying out theamplification reactions simultaneously. Thus, the reaction mixtureformed contained the test DNA genomic sample and the reference (wildtype) genomic DNA and all the primers, PX1, PX2, P2B, P2D, P1, and P3.This simplified protocol permitted the distinction between mutant orwild type DNA. In this procedure the initial PCR, step 1, and the secondPCR, step 2, were carried out with thermal cycling as described inExample 3, above. As in Example 3, branch migration was accomplished bya final denaturation at 94° C. for 1 min and annealing at 65° C. for 30minutes. The association of biotin and digoxin was detected by using thesignal producing system as in Example 1. Typical results from the assaycarried out by this protocol, where 20 ng of test and 20 ng of referencegenomic DNA per 50 μl reaction were co-amplified in the same tube, wereas follows (Table 5): TABLE 5 Sample Signal (RLU) blank  8046 WThomozygote (+/+)  12264 G542X/G542X homozygote (−/−) 167770 G542X/WTheterozygote (+/−) 102920

[0252] Note that all the steps in this protocol, except the detectionstep, were carried out in a single tube.

Example 5

[0253] In this example two commercially available thermostablepolymerases, Pfu DNA polymerase (Stratagene, La Jolla Calif.) and TaqDNA polymerase (Perkin Elmer, Norwalk Conn.) were utilized. The fidelityof Pfu polymerase (defined as the number of errors per nucleotide perPCR cycle) is known to be 12 times greater than the fidelity of Taqpolymerase due to the absence of 3′-5′ proofreading exonuclease activityin the latter enzyme (Lundberg, et al. (1991) Gene 108:1-6).

[0254] The simplified step-wise protocol utilized in Example 2 above wasfollowed with the exception that no wild type probe was necessary sincethe interest here was in detecting the heterozygotes with one wild typeand one mutant allele.

[0255] In the 1 st PCR, step 1, of the protocol genomic DNAs (40 ng per50 μL reaction volume) were amplified with the following primers for thepurpose of preparing amplicons for this example:

[0256] Primer PX2′: 5′-CAACTGTGGTTAAAGCAATAGTGT-3′ (SEQ ID NO: 7)

[0257] Primer PX1′: 5′-GCACAGATTCTGAGTAACCATAAT-3′ (SEQ ID NO: 8)

[0258] Both primers were from Oligos Etc., Inc., Wilsonville, Oreg. Theamplifications were carried out in a 96-well block of a UNO thermocyclerfrom Biometra, Tampa Fla.

[0259] After the initial denaturation step (95° C. for 4 min), 35 cycleswere performed consisting of 94° C. for 30 sec, 58° C. for 1 min and 72°C. for 1 min.

[0260] The resulting 425 bp amplicons were diluted 1:1000, and 1 μl (per50 μl reaction volume) aliquots of these dilutions were amplified in the2nd PCR, step 2 (20 cycles under the same conditions as in step 1) usingthe mixture of primers P2B, P2D, P1 and P3 identified above. 0.5 unitsof either Pfu DNA polymerase or Taq DNA polymerase per 50 μl were usedin both step 1 and step 2.

[0261] In Step 3 (branch migration), the samples were heated at 95° C.for 1 min. (denaturation) followed by 30 min. at 65° C. Step 4(detection) was performed exactly as described in Example 1 above.

[0262] The results are summarized in Table 6 below: TABLE 6 Pfu TaqDiscrim- Discrim- Signal ination Signal ination Sample (RLU) factor*(RLU) factor* blank   8606  21772 WT1 (+/+)  25014 1.00  125680 1.00 WT2(+/+)  24968 1.00  127468 1.02 WT3 (+/+)  28544 1.22  106574 0.83 WT4(+/+)  25982 1.06  125524 1.00 WT5 (+/+)  25206 1.01  119428 0.94G542X/G542X (−/−)  24996 1.00  245542 2.15 G542X/WT (+/−) 1151520 69.66 889890 8.35 G551D/WT (+/−) 1124920 68.03 1007210 9.48 G553X/WT (+/−)1353640 81.97 1014520 9.55 G560T/WT (+/−) 1249860 75.65 1132780 10.69

[0263] The results in Table 6 show that a thermostable polymerase havinga 3′-5′ proofreading exonuclease activity is preferred in the presentmethod for preparing amplicons for the purpose of mutation detectionusing the branch migration assay conducted according to the aboveprotocol.

Example 6

[0264] This example describes using ELISA as an alternative method fordetection of the branch migration products.

[0265] The samples used were as in Example 2 (Table 3) above: wt1, 542Xheterozygote and 542X/542X homozygote.

[0266] The wells of streptavidin-coated microtiter plates (Reacti-Bind™,Pierce, Rockford, Ill.) were washed once with 300 μl 0.5% DPBS(Dulbecco's Phosphate Buffered Saline; 10×DPBS contains 2 mg/l KCl, 2mg/l KH₂PO₄, 80 mg/l NaCl and 21.6 mg/l Na₂HPO₄, 7 H₂O), 0.05% Tween 20.

[0267] The DNA samples (2 μl) after step 3 (Example 2) were added to thewells containing 300 μl 1×PBS (Phosphate Buffered Saline; 10×PBScontains 1.44 mg/l KH₂PO₄, 90 mg/l NaCl and 7.95 mg/l Na₂HPO₄), 300 mMNaCl, 0.1% Tween 20, 25% Fetal Calf Serum, 3% BSA, 50 μg/ml sonicateddenatured calf thymus DNA. After incubation at 37° C. for 1 hr, thewells were washed 4 times with 300 μl 0.5% DPBS-0.05% Tween 20. 100 μlof 1:1000 dilution of the anti-digoxygenin Fab fragment (horseradishperoxidase conjugate, catalog # 1207733, Boehringer Mannheim,Indianapolis, Ind.) in the same buffer was added and the plate wasincubated at 37° C. for 1 hr. The wells were washed 4 times with 300 III0.5% DPBS-0.05% Tween 20. 100 μl of a 1:1 mixture of the TMB(tetramethylbenzidine) peroxidase substrate and H₂O₂ (Kirkegaard & PerryLaboratories, Gaithersburg, Md., product codes 50-76-01 and 50-65-00,respectively) was added and the plate was incubated at room temperaturefor 30 min. The color development was stopped by adding 100 μl 1 Mphosphoric acid and the signals were read using the Titertek MultiscanPlus microplate reader (ICN Biomedicals, Huntsville Ala.).

[0268] The results are summarized in Table 7 below: TABLE 7 Sample OD at450 nm Blank 0.065 WT homozygote (+/+) 0.134 G542X/G542X homozygote(−/−) 2.067 G542X/WT heterozygote (+/−) 1.036

Example 7

[0269] The protocol followed for this example was the simplifiedstep-wise protocol described in Example 2 above. In this example a 3-bpdeletion, ΔF508, in exon 10 (the most frequently occurring mutation) ofthe human cystic fibrosis gene (CFTR) was studied.

[0270] In the 1st PCR, step 1, human genomic DNA samples (50 ng) (fromRoche Molecular Systems, Alameda Calif., except for ΔF508/ΔF508homozygote(−/−), which was from Coriell Institute for Medical Research,Camden NJ) were amplified with the following primers:

[0271] Primer PX2″: 5′-CAAGTGAATCCTGAGCGTGA-3′ (SEQ ID NO: 9) and

[0272] Primer PX1″: 5′-CTAACCGATTGAATATGGAGCC-3′ (SEQ ID NO: 10).

[0273] Both primers were from Oligos Etc., Inc., Wilsonville, Oreg. Theamplification was carried out in a 96-well block of a UNO thermocyclerfrom Biometra, Tampa Fla. to generate a PCR product 340-bp in length.After the initial denaturation step (95° C. for 4 min), 35 cycles wereperformed consisting of 94° C. for 30 sec, 64° C. for 1 min and 72° C.for 1 min.

[0274] The resulting amplicons were diluted 1:1000, and 1 μl (per 50 μlreaction volume) aliquots of these dilutions were amplified in the 2ndPCR, step 2 (20 cycles under the same conditions as in step 1) using amixture of primers P2′B (or P2′D), P1′ and P3′. The resulting PCRproducts are 220-bp in length.

[0275] P2′: 5′-CTCAGTTTTCCTGGATTATGCC-3′ (SEQ ID NO: 11)

[0276] P2′D: digoxygenin-labeled P2′ from Genosys Biotechnologies, Inc.,Woodlands, Tex.

[0277] P2′B: biotinylated P2′ from Oligos Etc., Inc., Wilsonville, Oreg.

[0278] P1′: 5′-ACCATGCTCGAGATTACGAGCTAACCGATTGAATATGGAGCC-3′ (SEQ ID NO:12) from Oligos Etc., Inc., Wilsonville, Oreg.

[0279] P3′: 5′-GATCCTAGGCCTCACGTATTCTAACCGATTGAATATGGAGCC-3′, (SEQ IDNO: 13) from Oligos Etc., Inc., Wilsonville, Oreg.

[0280] Pa as part of primers P1′ and P3′ is identical to PX1″.

[0281] WT1 below was used as the reference sample and amplified withprimers P2′D, P1′ and P3′.

[0282] All the test samples were amplified with primers P2′B′, P1′ andP3′.

[0283] In step 3, branch migration, equal volumes of test and referenceamplicons were mixed and overlayed with mineral oil. The reactionmixture was heated for 1 min at 95° C. (denaturation) followed by 30 minat 65° C.

[0284] Step 4, detection, was carried out exactly as in Example 1. Theresults are summarized in the Table 8. TABLE 8 Sample Signal (RLU) Blank  4790 WT1 (+/+)  19834 WT2 (+/+)  18530 WT3 (+/+)  19496 WT4 (+/+) 19972 WT5 (+/+)  18460 WT6 (+/+)  19380 WT7 (+/+)  17980 ΔF508/ΔF508homozygote (−/−) 1341990 WT/ΔF508 heterozygote 1 (+/−)  524236 WT/ΔF508heterozygote 2 (+/−)  625440

Example 8 Simplified Direct Protocol

[0285] In this example the labeled and tailed amplicons for branchmigration were prepared directly from genomic DNA without a preliminaryamplification step. Accordingly, this example does not include the 1stPCR step (step 1 in the simplified step-wise protocol of Examples 2, 5,7) or the nested PCR using primers PX1 and PX2 (partial step-wiseprotocol of Example 3). Thus, the protocol was simplified further.

[0286] 50 ng of genomic DNA samples (as in Example 7) were amplifiedwith the primers P2′B (or P2′D), P1′ and P3′ (as in Example 7). 35cycles under the same cycling conditions as in step 2 of Example 7 wereperformed. Branch migration and detection steps were carried out exactlyas in Example 7.

[0287] The results are summarized in the Table 9. TABLE 9 Sample Signal(RLU) Blank   7696 WT1 (+/+)  34980 WT2 (+/+)  34790 WT3 (+/+)  35166WT4 (+/+)  32692 WT5 (+/+)  33846 WT6 (+/+)  38470 WT7 (+/+)  36374ΔF508/ΔF508 homozygote (−/−) 1824820 WT/ΔF508 heterozygote 1 (+/−) 447710 WT/ΔF508 heterozygote 2 (+/−)  812436

Example 9

[0288] In this example the simplified direct protocol described inExample 8 for the detection of the ΔF508 3-bp deletion was applied tothe 4 point mutations in exon 11. Two different pairs of labeled andtailed primers were used to prepare amplicons for branch migrationdirectly from genomic DNA.

[0289] Primer P2″: 5′-TAGAAGGAAGATGTGCCTTTCA-3′ (SEQ ID NO: 14)

[0290] P2″D and P2″B: digoxygenin and biotin-labeled P2″, respectively.

[0291] Primer P1″: 5′-ACCATGCTCGAGATTACGAGTTCTTAACCCACTAGCCATAAA-3′ (SEQID NO: 15)

[0292] Primer P3″: 5′-GATCCTAGGCCTCACGTATTTTCTTAACCCACTAGCCATAAA-3′ (SEQID NO: 16)

[0293] Primer P2′″: 5′-TTACATTAGAAGGAAGATGTGCCT-3′ (SEQ ID NO: 17) P2′″Dand P2′″B: digoxygenin and biotin-labeled P2″, respectively.

[0294] Primer P1′″: 5′-ACCATGCTCGAGATTACGAGGTGATTCTTAACCCACTAGCCA-3′(SEQ ID NO: 18)

[0295] Primer P3′″: 5′-GATCCTAGGCCTCACGTATTGTGATTCTTAACCCACTAGCCA-3′(SEQ ID NO: 19)

[0296] All primers were from Oligos Etc., Inc., Wilsonville, Oreg.

[0297] PCR from genomic DNA, branch migration and detection were carriedout exactly as described in Example 8 (37 PCR cycles were performed).The resulting PCR products were 333 bp and 343 bp in length,respectively.

[0298] WT1 below was used as the reference sample and amplified withprimers P2″D, P1″ and P3″ or P2′″D, P1′″ and P3′″, respectively (rightand left column, respectively, in Table 10 below).

[0299] All the test samples were amplified with primers P2″B, P1″ andP3″ or P2′″B, P1′″ and P3′″, respectively (right and left column,respectively, in Table 10 below). TABLE 10 Sample Signal (RLU) Blank  7384   8396 WT1 (+/+)  45456  56210 WT2 (+/+)  52480  49174 WT3 (+/+) 65172  56992 WT4 (+/+)  30778  88682 WT5 (+/+)  71906  63398G542X/G542X (−/−) 1797530 1148180 G542X/WT (+/−)  695056  473342G551D/WT(+/−)  902458  499874 G553X/WT(+/−)  859416  571882G560T/WT(+/−) 1030630  587710

[0300] In another experiment, the test and the reference genomic DNAsamples were co-amplified with a mixture of primers P2″B, P2″D, P1″ andP3″ (as in the Partial Step-Wise Approach of Example 3, only without anyouter primers). The results are summarized in Table 11 below. TABLE 11Sample Signal (RLU) Blank 7384 WT1 (+/+) 18166 WT2 (+/+) 16462 WT3 (+/+)20282 WT4 (+/+) 19106 WT5 (+/+) 21790 G542X/G542X (−/−) 640182 G542X/WT(+/−) 265984 G551D/WT (+/−) 294094 G553X/WT (+/−) 302366 G560T/WT (+/−)336964

Example 10

[0301] This example describes the application of the branch migrationassay for colony screening in the in vitro mutagenesis experiments. Theassay provided a rapid identification of the mutant clones.

[0302]E. coli clones of bacterial glucose-6-phosphate dehydrogenase gene(G6PDH) were produced in a manner similar to that described in EuropeanPatent Application No. 94 923 147.6. Amplification directly frombacterial clones was performed as follows. A small amount of bacterialcells were picked with a toothpick, and a toothpick was swirled in 50 μlof the PCR reaction mixture. After incubation at 95° C. for 5 min tolyse the cells, 35 PCR cycles were carried out consisting of 30 sec at94° C., 1 min at 66° C. and 1 rhin at 72° C. The resulting PCR productwas 320 bp in length. The PCR primers were as follows:

[0303] Primer P2″″: 5′-GTGTGGAATTGTGAGCGGATAA-3′ (SEQ ID NO: 20)

[0304] P2″″D and P2″″B: digoxygenin and biotin-labeled P2″″,respectively.

[0305] Primer P1″″: 5′-ACCATGCTCGAGATTACGAGGTGTGCACGGTATGAGAAATGT-3′(SEQ ID NO: 21)

[0306] Primer P3″″: 5′-GATCCTAGGCCTCACGTATTGTGTGCACGGTATGAGAAATGT-3′(SEQ ID NO: 22)

[0307] All primers were from Oligos Etc., Inc., Wilsonville, Oreg.

[0308] The reference amplicon was prepared from the purified dilutedplasmid containing the wild type G6PDH gene using the primers P2″″D,P1″″ and P3″″. The primers P2″″B, P1″″ and P3″″ were utilized in thecolony-PCR to prepare the test amplicons. The test and the referenceamplicons were mixed and subjected to branch migration and detection asdescribed in the examples above.

[0309] The results of the colony screening are summarized in Table 12below. TABLE 12 Clone Signal (RLU) Blank 7518 WT1 202084 WT2 300284 WT3337380 Mutant 1 (GA > TG) 1345450 Mutant 2 (AAA > TGC) 1464900 Mutant 3(GA > TG) 2106950

Example 11

[0310] This example describes an assay for the detection of a mutationin a nucleic acid using a set of four universal primers, two of each are5′-biotin (B)- and digoxigenin (D)-labeled, respectively. These primerswere the same as the following primers from Example 10 above: PrimerP2″″ (SEQ ID NO: 20), P2″″D and P2″″B: digoxygenin and biotin-labeledP2″″, respectively, Primer P1″″ (SEQ ID NO: 21) and Primer P3″″ (SEQ IDNO: 22).

[0311] Two sets of adapter primers, for exon 10 (ADF10 and ADR10),primers AP1 and AP2, respectively, and exon 11 (ADF11 and ADR11),primers AP3 and AP4, respectively, of the cystic fibrosis gene (fromgenomic DNA samples, Roche Molecular Systems, Alameda Calif.) were used.These adapter primers were as follows:

[0312] AP1: 5′ +E,uns GTGTGGAATTGTGAGCGGATAATAGAAGGAAGATGTGCCTTTCA 3′(SEQ ID NO: 23) wherein the underlined portion corresponds to P2″″ andthe non-underlined portion corresponds to P2″ (SEQ ID NO: 14) (Example9)

[0313] AP2: 5′ +E,uns GTGTGCACGGTATGAGAAATGTTTCTTAACCCACTAGCCATAAA 3′(SEQ ID NO: 24) wherein the underlined portion corresponds to the3′-region (nucleotides 21-42) of primers P1″″ and P3″″ and thenon-underlined portion corresponds to the 3′-region (nucleotides 21-42)of primers P1″ (SEQ ID NO: 15) (Example 9) and of P3″ (SEQ ID NO: 16)(Example 9)

[0314] AP3: 5′ +E,uns GTGTGGAATTGTGAGCGGATAACTCAGTTTTCCTGGATTATGCC 3′(SEQ ID NO: 25) wherein the underlined portion corresponds to P2″″ andthe non-underlined portion corresponds to P2′ (SEQ ID NO: 11) (Example7)

[0315] AP4: 5′ +E,uns GTGTGCACGGTATGAGAAATGTCTAACCGATTGAATATGGAGCC 3′(SEQ ID NO: 26) wherein the underlined portion corresponds to the3′-region (nucleotides 21-42) of primers P1″″ and P3″″ and thenon-underlined portion corresponds to PX1″ (SEQ ID NO: 10) (Example 11)

[0316] The initial amplifications with the adapter primers were carriedout in a 96-well block of a UNO thermocycler from Biometra, Tampa Fla.After the initial denaturation step (95° C. for 4 min), 30 cycles wereperformed consisting of 94° C. for 30 sec, 64° C. for 1 min and 72° C.for 1 min.

[0317] The resulting 244 bp (exon 10) and 357 bp (exon 11) ampliconswere diluted 1:1000, and 1 μl (per 50 μl reaction volume) aliquots ofthese dilutions were amplified in a second PCR step (step 2) (20 cyclesunder the same conditions as in step 1) using the mixture of primersP2″″B, P2″″D, P1″″ and P3″″ identified above. 0.625 units of either PfuDNA polymerase per 50 μl were used in both step 1 and step 2. Theresulting PCR products were a 264 bp (exon 10) and 377 bp (exon 11)amplicon, respectively.

[0318] In Step 3 (branch migration), the samples were heated at 95° C.for 1 min. (denaturation) followed by 30 min. at 65° C. Step 4(detection) was performed exactly as described in Example 1 above.

[0319] As a control, the primers specific for both amplicons were usedin the second PCR as follows: exon 10—P2′B, P2′D, P1′ and P3′ (Example8) and exon 11—P2″B, P2″D, P1″ and P3″ (Example 9). ΔF is ΔF508, 542 isG542X, 551 is G551 D, 553 is R553X and 560 is R560T. Both exon 10 andexon 11 mutations were successfully detected using this procedure. Theresults are summarized in Table 12. TABLE 12 Signal (RLU) exon 10 exon11 sample universal specific universal specific WT1 85404 59812 4104423726 WT2 58250 32162 41242 21204 WT3 60464 30446 49078 21510 ΔF/ΔF65640 42086 39790 18926 542/542 64666 32188 73300 39870 WT/542 6271039394 531954 751084 WT/551 56124 26768 823282 1100860 WT/553 58550 28060547434 888654 ΔF/560 629520 804126 597640 846434

Example 12

[0320] An equimolar mixture of the following primers (totalconcentration of each forward and reverse primers 250 nM) was used.

[0321] Unmodified Primers:

[0322] The unmodified primers used in this Example 12 were described inExample 1 as follows:

[0323] P2B: 5′GCCTTTCAAATTCAGATTGAGC 3′ (SEQ ID NO: 3) (P2) biotinylatedat the 5′-end

[0324] P2D: forward primer P2 labeled with digoxygenin at the 5′-end

[0325] P1: a reverse primer having a 3′-portion identical with Pa (SEQID NO: 4) and an additional 5′-“tail” t1 (underlined) 20 nucleotideslong:

[0326] 5′-+E,uns ACCATGCTCGAGATTACGAGGACATTTACAGCAAATGCTTGC-3′ (SEQ IDNO: 5)

[0327] P3: reverse primer having a 3′-portion identical with Pa and anadditional 5′-“tail” t2 (underlined) 20 nucleotides long:

[0328] 5′-+E,uns GATCCTAGGCCTCACGTATTGACATTTACAGCAAATGCTTGC-3′ (SEQ IDNO: 6)

[0329] 3′-etheno Modified Oligonucleotides (Modified Oligos):

[0330] P2cB, P2cD, P1c and P3c are the same as P2B, P2D, P1 and P3 butwith two etheno-dA's followed by unmodified dG at their 3′-ends. Theseoligonucleotides were also purchased from Oligos, Etc.

[0331] P1c: 5′-GCCTTTCAAATTCAGATTGAGC-NN-G 3′ where X is etheno-deoxy A(SEQ ID NO: 27)

[0332] P2cB: 5′GCCTTTCAAATTCAGATTGAGC-NN-G 3′ where X is etheno-deoxy A(SEQ ID NO: 28) biotinylated at the 5′-end

[0333] P2cD: 5′GCCTTTCAAATTCAGATTGAGC-NN-G 3′ where X is etheno-deoxy A(SEQ ID NO: 29) biotinylated at the 5′-end5′-GATCCTAGGCCTCACGTATTGACATTTACAGCAAATGCTTGC-NN-G 3′ where X isetheno-deoxy A (SEQ ID NO: 30)

[0334] Thirty five PCR cycles (30 sec at 94° C., 1 min at 64° C., 1 minat 72° C.) were performed using 50 ng genomic DNA and 0.625 U Pfupolymerase per 50 μl. The hot start procedure using PCR GEM50 wax beads(Perkin Elmer, Norwalk Conn.) was utilized.

[0335] Branch Migration:

[0336] After completion of PCR as described above, the samples weresubjected to branch migration: 95° C., 1 min (denaturation), followed by65° C., 30 min (branch migration).

[0337] Detection:

[0338] Detection was carried out in a manner similar to that describedin U.S. Pat. No. 5,340,716, the relevant portions of which areincorporated herein by reference. The procedure and amounts of thecomponents were as follows:

[0339] A 2 μl aliquot of the reaction mixture from the above branchmigration was mixed with 100 μl of the beads suspension (2.5 μg ofSens-Sav beads and 2.5 μg Acc-Ab_(Dig) beads per 100 μl buffer A),incubated at 37° C. for 5 min. The reaction mixture was then irradiatedwith a 150 watt Xenon lamp for 3 sec (3 cycles of 1 sec illumination and1 sec waiting time) and the signal was then read.

[0340] The results of a typical experiment conducted according to theprotocol of this Example 12 were as follows (Table 13): TABLE 13 ControlInvention Unmodified primers Modified oligos Sample Signal (RLU) Signal(RLU) WT1 71252 6522 WT2 75088 6186 WT3 80016 7634 WT4 80052 6998G542X/WT heterozygote 235644 576452 G551D/WT heterozygote 193542 426382R553X/WT heterozygote 158058 390062 R560T/WT heterozygote 187078 440826

[0341] WT1-4 are wild type homozygotes.

[0342] The results obtained demonstrate that the use of modifiedoligonucleotide primers that contain one or more unnatural nucleotidesat the 3′-end that do not hybridize to the target sequence, e.g.,3′-etheno modified oligonucleotides, in an assay in accordance with thepresent invention resulted in an improvement over the same assay usingunmodified primers. The wild type signal decreased by an order ofmagnitude whereas the mutant signal increased by approximately a factorof two with the average signal-to-background ratio increasing from 2.5(marginal) to 67. Other modified oligonucleotides to be used include,for example, modified oligonucleotides5′-GCCTTTCAAATTCAGATTGAGC-NN-G-3′, where X is:

[0343] a) O-6-methyl deoxy G (P1a) (SEQ ID NO: 31)

[0344] b) O-4-methyl deoxy T (P1b) (SEQ ID NO: 32)

Example 13

[0345] In Example 12, the 3′-etheno modified oligonucleotides were aidedby implementation of the standard hot start procedure utilizing waxbeads. In this Example 13 the hot start procedure employed in Example 12was not used. PCR conditions were the same as in Example 12, except thatall components of the PCR reactions were mixed together at roomtemperature or on ice. The results are summarized in Table 14. TABLE 14Signal (RLU) Signal (RLU) RT Ice Control Invention Control InventionUnmodified Modified Unmodified Modified Sample primers oligos primersoligos WT1 97992 14988 112050 8612 WT2 112110 10566 100784 9634 G551D/WT59828 49910 99516 101016 R553X/WT 51640 45180 90592 121808

[0346] When the reactions were assembled at room temperature, theaverage signal to background ratio of 5.3 was observed with the3′-etheno modified oligonucleotides as compared to no discriminationbetween mutant and wild type samples with the unmodified primers (Table14, left). When the reactions were assembled on ice, the average signalto background ratio increased to 24.2 for the 3′-etheno primers, whereasstill no discrimination was observed for the unmodified primers (Table14, right).

[0347] The above results demonstrated that the 3′-5′ exonucleaseactivity of the Pfu polymerase was occurring at room temperature andremoved the modified nucleotides from some of the molecules of themodified oligonucleotides before PCR cycling began. To slow this processdown, smaller amounts of Pfu polymerase were used. In order to preservethe efficiency and yield of PCR, Pfu polymerase was supplemented withPfu exo- (from Stratagene, La Jolla Calif.) in which the 3′-5′exonuclease activity is missing. PCR conditions were the same as above,except that 40 cycles were performed. The total amount of polymerase(Pfu+Pfu exo⁻) per reaction was 0.625 U. To make the conditionsmaximally unfavorable (to encourage non-specific priming), PCR reactionscontaining 3′-etheno modified oligonucleotides were left at RT for aslong as 30 min prior to starting thermocycling. The results aresummarized in Table 15. TABLE 15 Signal (RLU) Pfu exo⁻/Pfu Pfu exo⁻/PfuSample Pfu (2:1) (1:1) WT1 18970 8704 14540 WT2 18652 9234 21656G551D/WT 103236 173964 314368 R553X/WT 75130 210706 337192

[0348] The results summarized in Table 15 demonstrated that, when themixture of two enzymes (Pfu and Pfu exo⁻) was used, the 3′-ethenoprimers were very effective as judged by a signal to background ratio ofabout 20 as compared with a signal to background ratio of about 4.5 forPfu alone.

[0349] By employing the method of the present invention in conjunctionwith the use of modified oligonucleotide primers, an improvement of thesignal-to-background ratio in a direct approach as applied to thedetection of 4 point mutations in exon 11 of the human CFTR gene wasrealized as demonstrated by this Example 13.

[0350] The above discussion includes certain theories as to mechanismsinvolved in the present invention. These theories should not beconstrued to limit the present invention in any way, since it has beendemonstrated that the present invention achieves the results described.

[0351] The above description and examples fully disclose the inventionincluding preferred embodiments thereof. Modifications of the methodsdescribed that are obvious to those of ordinary skill in the art such asmolecular biology and related sciences are intended to be within thescope of the claims.

1 32 33 base pairs nucleic acid single linear DNA (genomic) NO NOC-terminal not provided 1 CAACTGTGGT TAAAGCAATA GTGTGATATA TGA 33 32base pairs nucleic acid single linear DNA (genomic) NO NO C-terminal notprovided 2 GCACAGATTC TGAGTAACCA TAATCTCTAC CA 32 22 base pairs nucleicacid single linear DNA (genomic) NO NO C-terminal not provided 3GCCTTTCAAA TTCAGATTGA GC 22 22 base pairs nucleic acid single linear DNA(genomic) NO NO C-terminal not provided 4 GACATTTACA GCAAATGCTT GC 22 42base pairs nucleic acid single linear DNA (genomic) NO NO C-terminal notprovided 5 ACCATGCTCG AGATTACGAG GACATTTACA GCAAATGCTT GC 42 42 basepairs nucleic acid single linear DNA (genomic) NO NO C-terminal notprovided 6 GATCCTAGGC CTCACGTATT GACATTTACA GCAAATGCTT GC 42 24 basepairs nucleic acid single linear DNA (genomic) NO NO C-terminal notprovided 7 CAACTGTGGT TAAAGCAATA GTGT 24 24 base pairs nucleic acidsingle linear DNA (genomic) NO NO C-terminal not provided 8 GCACAGATTCTGAGTAACCA TAAT 24 20 base pairs nucleic acid single linear DNA(genomic) NO NO C-terminal not provided 9 CAAGTGAATC CTGAGCGTGA 20 22base pairs nucleic acid single linear DNA (genomic) NO NO C-terminal notprovided 10 CTAACCGATT GAATATGGAG CC 22 22 base pairs nucleic acidsingle linear DNA (genomic) NO NO C-terminal not provided 11 CTCAGTTTTCCTGGATTATG CC 22 42 base pairs nucleic acid single linear DNA (genomic)NO NO C-terminal not provided 12 ACCATGCTCG AGATTACGAG CTAACCGATTGAATATGGAG CC 42 42 base pairs nucleic acid single linear DNA (genomic)NO NO C-terminal not provided 13 GATCCTAGGC CTCACGTATT CTAACCGATTGAATATGGAG CC 42 22 base pairs nucleic acid single linear DNA (genomic)NO NO C-terminal not provided 14 TAGAAGGAAG ATGTGCCTTT CA 22 42 basepairs nucleic acid single linear DNA (genomic) NO NO C-terminal notprovided 15 ACCATGCTCG AGATTACGAG TTCTTAACCC ACTAGCCATA AA 42 42 basepairs nucleic acid single linear DNA (genomic) NO NO C-terminal notprovided 16 GATCCTAGGC CTCACGTATT TTCTTAACCC ACTAGCCATA AA 42 24 basepairs nucleic acid single linear DNA (genomic) NO NO C-terminal notprovided 17 TTACATTAGA AGGAAGATGT GCCT 24 42 base pairs nucleic acidsingle linear DNA (genomic) NO NO C-terminal not provided 18 ACCATGCTCGAGATTACGAG GTGATTCTTA ACCCACTAGC CA 42 43 base pairs nucleic acid singlelinear DNA (genomic) NO NO C-terminal not provided 19 GATCCTAGGCCTCACGTATT GTGATTACTT AACCCACTAG CCA 43 22 base pairs nucleic acidsingle linear DNA (genomic) NO NO C-terminal not provided 20 GTGTGGAATTGTGAGCGGAT AA 22 42 base pairs nucleic acid single linear DNA (genomic)NO NO C-terminal not provided 21 ACCATGCTCG AGATTACGAG GTGTGCACGGTATGAGAAAT GT 42 42 base pairs nucleic acid single linear DNA (genomic)NO NO C-terminal not provided 22 GATCCTAGGC CTCACGTATT GTGTGCACGGTATGAGAAAT GT 42 44 base pairs nucleic acid single linear DNA (genomic)NO NO C-terminal not provided 23 GTGTGGAATT GTGAGCGGAT AATAGAAGGAAGATGTGCCT TTCA 44 44 base pairs nucleic acid single linear DNA(genomic) NO NO C-terminal not provided 24 GTGTGCACGG TATGAGAAATGTTTCTTAAC CCACTAGCCA TAAA 44 44 base pairs nucleic acid single linearDNA (genomic) NO NO C-terminal not provided 25 GTGTGGAATT GTGAGCGGATAACTCAGTTT TCCTGGATTA TGCC 44 44 base pairs nucleic acid single linearDNA (genomic) NO NO C-terminal not provided 26 GTGTGCACGG TATGAGAAATGTCTAACCGA TTGAATATGG AGCC 44 25 base pairs nucleic acid single linearDNA (genomic) NO NO C-terminal not provided 27 GCCTTTCAAA TTCAGATTGAGCNNG 25 25 base pairs nucleic acid single linear DNA (genomic) NO NOC-terminal not provided 28 GCCTTTCAAA TTCAGATTGA GCNNG 25 25 base pairsnucleic acid single linear DNA (genomic) NO NO C-terminal not provided29 GCCTTTCAAA TTCAGATTGA GCNNG 25 45 base pairs nucleic acid singlelinear DNA (genomic) NO NO C-terminal not provided 30 GATCCTAGGCCTCACGTATT GACATTTACA GCAAATGCTT GCNNG 45 25 base pairs nucleic acidsingle linear DNA (genomic) NO NO C-terminal not provided 31 GCCTTTCAAATTCAGATTGA GCNNG 25 25 base pairs nucleic acid single linear DNA(genomic) NO NO C-terminal not provided 32 GCCTTTCAAA TTCAGATTGA GCNNG25

What is claimed is:
 1. A method for detecting the presence of adifference between two related nucleic acid sequences, said methodcomprising: (a) forming a complex comprising both of said nucleic acidsequences in double stranded form, wherein each member of at least onepair of non-complementary strands within said complex has a label and(b) detecting the association of said labels within said complex, theassociation thereof being related to the presence of said difference. 2.The method of claim 1 wherein said difference is a mutation.
 3. Themethod of claim 1 wherein said nucleic acid sequences are DNA.
 4. Themethod of claim 1 wherein said complex comprises a Holliday junction. 5.A method for detecting a mutation within a target nucleic acid sequence,said method comprising: (a) forming from said target sequence a tailedtarget partial duplex A′ comprised of a duplex of said target sequence,a label and at one end of said duplex, two non-complementaryoligonucleotides, one linked to each strand, (b) providing incombination said tailed target partial duplex A′ and a tailed referencepartial duplex B′ lacking said mutation having a label as a partthereof, wherein said tailed reference partial duplex B′ is comprised oftwo nucleic acid strands that are complementary to the strands in saidtailed target partial duplex A′ but for the possible presence of amutation and wherein said labels are present in non-complementarystrands of said tailed target and tailed reference partial duplexesrespectively, and (c) detecting, by means of said labels, the formationof a complex between said tailed partial duplexes, the formation thereofbeing directly related to the presence of said mutation.
 6. The methodof claim 5 wherein said target nucleic acid sequence is DNA.
 7. Themethod of claim 5 wherein said tailed reference partial duplex B′ isprovided in said combination by forming said tailed reference partialduplex B′ in the same reaction medium as that used for step (a).
 8. Themethod of claim 7 wherein forming said tailed target partial duplex A′and said tailed reference partial duplex B′ is carried outsimultaneously.
 9. The method of claim 5 wherein said labels areindependently selected from the group consisting of oligonucleotides,enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes,enzyme substrates, radioactive groups, small organic molecules and solidsurfaces.
 10. The method of claim 6 wherein said non-complementaryoligonucleotides each have from 15 to 60 nucleotides.
 11. A method ofdetecting a mutation within a target nucleic acid sequence, said methodcomprising: (a) amplification of said target sequence by polymerasechain reaction, using primers P1 and P2 to produce an amplicon AA,wherein one of said primers P1 and P2 comprises a label and wherein saidprimer P1 is comprised of a 3′-end portion Pa that can hybridize withsaid target sequence and 5-end portion B1 that cannot hybridize withsaid target sequence, (b) extending a primer P3 by chain extension alongone strand of amplicon AA to produce a tailed target partial duplex A′,wherein said primer P3 is comprised of said 3′-end portion Pa and a5′-end portion A1 that cannot hybridize to said target sequence or itscomplement, (c) amplification of a reference nucleic acid sequence,using said primer P2 and said primer P3, by polymerase chain reaction toproduce amplicon BB, said reference sequence being identical to saidtarget sequence but lacking a possible mutation, wherein said primer P2comprises a label when said primer P2 in step (a) above comprises alabel and said primer P3 comprises a label when said primer P1 in step(a) above comprises a label, (d) extending said primer P1 by chainextension along one strand of amplicon BB to produce a tailed referencepartial duplex B′, (e) allowing said tailed target partial duplex A′ tobind to said tailed reference partial duplex B′, and (f) detecting thebinding of one of said labels to another of said labels as a result ofthe formation of a complex between said tailed partial duplexes, thebinding thereof being directly related to the presence of said mutation.12. The method of claim 11 wherein said amplification of step (c) iscarried out in the same reaction medium as that used for step (a). 13.The method of claim 12 wherein said amplification of step (c) is carriedout simultaneously with the amplification of step (a).
 14. The method ofclaim 11 wherein the label of primer P2 in step (c) is different thanthe label of primer P2 in step (a).
 15. The method of claim 14 whereinsaid labels are independently selected from the group consisting ofoligonucleotides, enzymes, dyes, fluorescent molecules,chemiluminescers, coenzymes, enzyme substrates, radioactive groups,small organic molecules and solid surfaces.
 16. The method of claim 14wherein said nucleic acid is DNA.
 17. A method for detecting a mutationin a nucleic acid, said method comprising: (a) producing, from a targetnucleic acid sequence suspected of having a mutation, a partial duplexA′ comprising a fully complementary double stranded nucleic acidsequence containing said target nucleic acid sequence wherein one strandhas at its 5′-end a portion A1 that does not hybridize with acorresponding portion A2 at the 3′-end of the other strand, wherein oneof said strands of said partial duplex A′ comprises a label, (b)producing, from a reference nucleic acid sequence that corresponds tosaid target nucleic acid sequence of step (a) except for said mutation,a partial duplex B′ comprising said double stranded nucleic acidsequence lacking said mutation wherein the strand corresponding to thestrand comprising said portion A1 has at its 5′-end a portion B1 that iscomplementary with said A2 and the other strand has at its 3′-end aportion B2 that is complementary with said A1, wherein one of saidstrands of said partial duplex B′ comprises a label, said strandcomprising said label being unable to hybridize directly to said strandof said partial duplex A′ that comprises a label, (c) subjecting saidpartial duplexes A′ and B′ to conditions that permit said duplexes tohybridize to each other wherein, if said target nucleic acid sequencehaving said mutation is present, a stable complex is formed comprisingsaid partial duplex A′ and said partial duplex B′, and (d) determiningwhether said stable complex is formed, the presence thereof indicatingthe presence of said nucleic acid having said mutation.
 18. The methodof claim 17 wherein said labels are independently selected from thegroup consisting of oligonucleotides, enzymes, dyes, fluorescentmolecules, chemiluminescers, coenzymes, enzyme substrates, radioactivegroups, small organic molecules, polynucleotide sequences and solidsurfaces.
 19. The method of claim 17 wherein steps (a) and (b) arecarried out simultaneously in the same reaction medium.
 20. The methodof claim 17 wherein said A1 and said A2 each have from 15 to 60nucleotides.
 21. The method of claim 17 wherein said nucleic acid isDNA.
 22. A method for detecting the presence of a difference between tworelated nucleic acid sequences, said method comprising: (a) treating amedium suspected of containing said two related nucleic acid sequencesto provide two partial duplexes each comprised of duplexes having at oneend therein non-complementary end portions thereby forming two partialduplexes, wherein said partial duplexes are related in that, except forsaid difference, one of the strands S1 of one of said partial duplexesis complementary to one of the strands S1′ of the other of said partialduplexes and the other of the strands S2 of said one of said partialduplexes is complementary to the other of the strands S2′ of said otherof said partial duplexes, (b) subjecting said medium to conditions thatpermit the binding of S1 to S1′ and S2 to S2′, respectively, wherein, ifthere is a difference between said related nucleic acid sequences, astable complex is formed comprising said strands Si, Si′, S2 and S2′,and (c) determining whether said stable complex is formed, the presencethereof indicating the presence of a difference between said relatednucleic acid sequences.
 23. The method of claim 22 whereinnon-complementary strands within said complex have labels.
 24. Themethod of claim 23 wherein the association of said labels as part ofsaid complex is detected, the association thereof being related to thepresence of said difference.
 25. The method of claim 23 wherein saidlabels are independently selected from the group consisting ofoligonucleotides, enzymes, dyes, fluorescent molecules,chemiluminescers, coenzymes, enzyme substrates, radioactive groups,small organic molecules, polynucleotide sequences and solid surfaces.26. A method of preparing a DNA partial duplex having a portion at anend thereof that has two predefined non-complementary single strandedsequences, said method comprising: (a) combining a medium containing anucleic acid with a polymerase, nucleoside triphosphates and twoprimers, wherein one of said primers P3 is extendable along one of saidstrands of said nucleic acid, said P3 having a 3′-end portion Pa thatdoes bind and a 5′-end portion A1 that does not bind thereto, and theother of said primers P2 is extendable along the other of said strandsof said nucleic acid, wherein the extended primer produced by theextension of one of said primers is a template for the other of saidprimers, (b) subjecting said medium to temperature cycling to extendsaid primers, and (c) combining said medium with a primer P1 whereinsaid P1 has said 3′-end portion Pa and a 5′-end portion B1 that does notbind to said extended P2 primer, and (d) subjecting said medium toconditions such that said P1, binds to and is extended along saidextended primer P2 to produce only a complement, and not a copy, of saidextended primer.
 27. The method of claim 26 wherein a label is bound toP2 or P1.
 28. The method of claim 27 wherein said labels areindependently selected from the group consisting of oligonucleotides,enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes,enzyme substrates, radioactive groups, small organic molecules,polynucleotide sequences and solid surfaces.
 29. A method of preparing aDNA partial duplex having a portion at one end that has twonon-complementary single stranded sequences, said method comprising: (a)combining a medium containing a single stranded polynucleotide with aprimer P1 wherein said P1 has a 3′-end portion Pa that binds to asequence that is 8 to 60 nucleotides from the 3′-end of said singlestranded polynucleotide and a 8 to 60 nucleotide portion B1 that doesnot bind to said single stranded polynucleotide, and (b) subjecting saidmedium to conditions under which P1, binds to and is extended along saidsingle stranded polynucleotide.
 30. The method of claim 29 wherein saidP1 comprises a label.
 31. The method of claim 30 wherein said labels areindependently selected from the group consisting of oligonucleotides,enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes,enzyme substrates, radioactive groups, small organic molecules,polynucleotide sequences and solid surfaces.
 32. A kit for detecting amutation in a target nucleic acid, said kit comprising in packagedcombination: (a) a primer P2 that is extendable along one of saidstrands of said target nucleic acid, (b) a primer P1 comprising a 3′-endportion Pa that binds to, and is extendable along, the other of saidstrands of said target nucleic acid and a 5′-end portion B1 that doesnot bind to said target nucleic acid, and (c) a primer P3 comprisingsaid 3′-end portion Pa and a portion A1 that is different than said B1and does not bind to said target nucleic acid.
 33. The kit of claim 32which comprises a reference nucleic acid.
 34. The kit of claim 32 whichcomprises: (a) a polymerase, (b) nucleoside triphosphates, and (c) apair of primers for amplifying said target and said reference nucleicacids.
 35. The kit of claim 34 wherein all of said components arepackaged in the same container.
 36. The kit of claim 32 wherein at leastone of said primers comprises a label.
 37. The kit of claim 36 whereinsaid labels are independently selected from the group consisting ofoligonucleotides, enzymes, dyes, fluorescent molecules,chemiluminescers, coenzymes, enzyme substrates, radioactive groups,small organic molecules, polynucleotide sequences and solid surfaces.38. A method for detecting a difference between two related nucleic acidsequences, said methods comprising: (a) forming a quadramolecularcomplex comprising both of said nucleic acid sequences in doublestranded form and (b) detecting the presence of said complex by bindingsaid complex to a receptor, the presence of said complex indicating thepresence of a difference between said sequences.
 39. A method fordetecting a target nucleic acid sequence, said method comprising: (a)forming from said target sequence a tailed target partial duplex A′comprised of a duplex of said target sequence, a label, and at one endof said duplex, two non-complementary oligonucleotides, one linked toeach strand, (b) providing in combination (i) said tailed target partialduplex A′ and (ii) a tailed reference partial duplex B′ comprising aduplex of a sequence different than said target sequence, a label and,at one end of said duplex, two oligonucleotides that are complementaryto said two non-complementary oligonucleotides, one linked to eachstrand wherein said labels are on non-complementary strands, and (c)detecting, by means of said labels, the formation of a complex betweensaid partial duplexes A′ and B′, the formation thereof being directlyrelated to the presence of said target nucleic acid.
 40. The method ofclaim 39 wherein said target and said reference nucleic acid sequencesare identical but for a mutation.
 41. The method of claim 39 fordetecting a target nucleic acid sequence that does not contain amutation.
 42. A method of detecting a target nucleic acid sequence, saidmethod comprising: (a) amplification of said target sequence bypolymerase chain reaction, using primers P1 and P2 to produce anamplicon AA, wherein one of said primers P1 and P2 comprises a label andsaid primer P1 is comprised of a 3′-end portion Pa that can hybridizewith said target sequence and 5′-end portion B1 that cannot hybridizewith said target sequence, (b) extending a primer P3 by chain extensionalong one strand of amplicon AA to produce a tailed target partialduplex A′, wherein said primer P3 is comprised of said 3′-end portion Paand a 5′-end portion A1 that cannot hybridize to said target sequence orits complement, (c) amplification of a reference nucleic acid sequencedifferent than said target nucleic acid sequence, using said primer P2and said primer P3, by polymerase chain reaction to produce amplicon BB,wherein said primer P2 comprises a label when said primer P2 in step (a)above comprises a label and said primer P3 comprises a label when saidprimer P1 in step (a) above comprises a label, (d) extending said primerP1 by chain extension along one strand of amplicon BB to produce atailed reference partial duplex B′, (e) allowing said tailed targetpartial duplex A′ to bind to said tailed reference partial duplex B′ toform a complex, and (f) detecting the binding of one of said labels toanother of said labels as a result of the formation of said complex, thebinding thereof being directly related to the presence of said targetnucleic acid sequence.
 43. The method of claim 42 wherein said targetand said reference nucleic acid sequences are identical but for amutation.
 44. The method of claim 42 for detecting a target nucleic acidsequence that does not contain a mutation.
 45. A method for detecting atarget nucleic acid sequence, said method comprising: (a) producing,from a target nucleic acid sequence, a partial duplex A′ comprising afully complementary double stranded nucleic acid sequence containingsaid target nucleic acid sequence wherein one strand has at its 5′-end aportion A1 that does not hybridize with a corresponding portion A2 atthe 3′-end of the other strand, wherein one of said strands of saidpartial duplex A′ comprises a label, (b) producing, from a referencenucleic acid sequence, a partial duplex B′ comprising a double strandednucleic acid sequence different from said target nucleic acid sequence,wherein the strand corresponding to the strand comprising said portionA1 has at its 5′-end a portion B1 that is complementary with said A2 andthe other strand has at its 3′-end a portion B2 that is complementarywith said Al, wherein one of said strands of said partial duplex B′comprises a label, said strand comprising said label being unable tohybridize directly to said strand of said partial duplex that comprisesa label, (c) subjecting said partial duplexes A′ and B′ to conditionsthat permit said duplexes to hybridize to each other to form aquadramolecular complex, and (d) determining whether said complex isformed, the presence thereof indicating the presence of said targetnucleic acid sequence.
 46. The method of claim 45 wherein said targetand said reference nucleic acid sequences are identical but for amutation.
 47. The method of claim 45 for detecting a target nucleic acidsequence that does not contain a mutation.
 48. A method for detectingthe presence of a difference between two related nucleic acid sequences,said method comprising: (a) producing from said two related nucleic acidsequences a target nucleic acid sequence and a reference nucleic acidsequence wherein each respective strand of said target nucleic acidsequence has a portion introduced therein that is a nucleotide sequencepriming site and wherein each respective strand of said referencenucleic acid sequence has a portion introduced therein that is anucleotide sequence priming site, (b) producing, from said targetnucleic acid sequence using said nucleotide sequence priming sites, apartial duplex A′ comprising a fully complementary double strandednucleic acid sequence containing said target nucleic acid sequencewherein one strand has at its 5′-end a portion Al that does nothybridize with a corresponding portion A2 at the 3′-end of the otherstrand, (c) producing, from said reference nucleic acid sequence usingsaid nucleotide sequence priming sites, a partial duplex B′ comprisingsaid double stranded nucleic acid sequence wherein the strandcorresponding to the strand comprising said portion A1 has at its 5′-enda portion B1 that is complementary with said A2 and the other strand hasat its 3′-end a portion B2 that is complementary with said A1, (d)subjecting said partial duplexes A′ and B′ to conditions that permitsaid duplexes to hybridize to each other wherein, if said relatednucleic acid sequences have a difference, a stable complex is formedcomprising said partial duplex A′ and said partial duplex B′, and (e)determining whether said stable complex is formed, the presence thereofindicating the presence of said difference between said two relatednucleic acid sequences.
 49. The method of claim 48 wherein steps (a),(b) and (c) are carried by polymerase chain reaction and wherein saidpriming sites are introduced into each respective strand of said targetnucleic acid sequence and said reference nucleic acid sequence in step(a) oligonucleotide primers each having a 3′-end portion that hybridizesto a respective strand of said related nucleic acid sequences and aportion 5′ of said 3′-end portion that does not hybridize to said strandand is capable of hybridizing to a respective oligonucleotide primerused in step (b) or step (c).
 50. The method of claim 48 wherein one ofthe strands of said partial duplex A′ comprises a label and one of thestrands of said partial duplex B′ comprises a label wherein said strandcomprising said label is unable to hybridize directly to said strand ofsaid partial duplex A′ that comprises a label.
 51. The method of claim50 wherein said labels are independently selected from the groupconsisting of oligonucleotides, enzymes, dyes, fluorescent molecules,chemiluminescers, coenzymes, enzyme substrates, radioactive groups,small organic molecules, polynucleotide sequences and solid surfaces.52. The method of claim 48 wherein step (a) is carried out in a separatereaction container from that in which steps (b) and (c) are carried out.53. The method of claim 48 wherein said A1 and said A2 each have from 15to 60 nucleotides.
 54. The method of claim 48 wherein said nucleic acidis DNA.
 55. A method for detecting the presence of a mutation in atarget nucleic acid sequence, said method comprising: (a) amplificationof said target nucleic acid sequence by polymerase chain reaction usingprimers PX1 i and PX2 i to produce a target sequence comprisingnucleotide sequence priming sites Pa′ and P2′, (b) amplification of areference nucleic acid sequence by polymerase chain reaction usingprimers PX1 i and PX2 i to produce a reference sequence comprisingnucleotide sequence priming sites Pa′ and P2′, said reference sequencebeing identical to said target sequence but lacking a possible mutation,(c) amplification of said target sequence produced in step (a) bypolymerase chain reaction, using primers P1 and P2 to produce anamplicon AA, wherein said primer P1 is comprised of a 3′-end portion Pathat can hybridize with priming site Pa′ of said target sequence and5′-end portion B1 that cannot hybridize with said target sequence, (d)extending a primer P3 by chain extension along one strand of amplicon AAto produce a tailed target partial duplex A′, wherein said primer P3 iscomprised of said 3′-end portion Pa and a 5′-end portion A1 that cannothybridize to said target sequence or its complement, (e) amplificationof said reference sequence produced in step (b), using said primer P2and said primer P3, by polymerase chain reaction to produce amplicon BB,(f) extending said primer P1 by chain extension along one strand ofamplicon BB to produce a tailed reference partial duplex B′, (g)allowing said tailed target partial duplex A′ to bind to said tailedreference partial duplex B′, and (h) detecting the formation of acomplex between said tailed partial duplexes, the binding thereof beingdirectly related to the presence of said mutation.
 56. The method ofclaim 55 wherein said amplification of step (b) is carried out in thesame reaction medium as that used for step (a) and in a differentreaction medium from that used for steps (c) and (e).
 57. The method ofclaim 55 wherein said amplification of step (b) is carried outsimultaneously with the amplification of step (a).
 58. The method ofclaim 55 wherein in step (c) one of said primers P1 and P2 comprises alabel and wherein in step (e) said primer P2 comprises a label when saidprimer P2 in step (c) comprises a label and said primer P3 comprises alabel when said primer P1 in step (c) comprises a label.
 59. The methodof claim 58 wherein the label of primer P2 in step (e) is different thanthe label of primer P2 in step (c).
 60. The method of claim 59 whereinsaid labels are independently selected from the group consisting ofoligonucleotides, enzymes, dyes, fluorescent molecules,chemiluminescers, coenzymes, enzyme substrates, radioactive groups,small organic molecules and solid surfaces.
 61. The method of claim 59wherein said nucleic acid is DNA.
 62. A kit for detecting a mutation ina target nucleic acid, said kit comprising in packaged combination: (a)a primer P2 that is extendable along one of said strands of said targetnucleic acid, (b) a primer P1 comprising a 3′-end portion Pa that bindsto, and is extendable along, the other of said strands of said targetnucleic acid and a 5′-end portion B1 that does not bind to said targetnucleic acid, (c) a primer P3 comprising said 3′-end portion Pa and aportion A1 that is different than said B1 and does not bind to saidtarget nucleic acid, and (d) a pair of primers for amplifying saidtarget and said reference nucleic acids wherein one of said primers hasa 3′-end portion that is hybridizable to said target and said referencenucleic acids and a portion 5′ thereof that is not hybridizable withsaid target or said reference nucleic acids and is substantiallyidentical to said primer P2 and the other of said primers has a 3′-endportion that is hybridizable to said target and said reference nucleicacids and a portion 5′ thereof that is not hybridizable with said targetor said reference nucleic acids and is substantially identical to said3′-end portion Pa of said primers P1 and P3.
 63. The kit of claim 62which comprises a reference nucleic acid.
 64. The kit of claim 62 whichcomprises: (a) a polymerase and (b) nucleoside triphosphates.
 65. Thekit of claim 62 wherein components (a)-(c) are packaged in one containerand components (d) are packaged in a separate container.
 66. The kit ofclaim 62 wherein at least one of said primers P1, P2 or P3 comprises alabel.
 67. The kit of claim 66 wherein said labels are independentlyselected from the group consisting of oligonucleotides, enzymes, dyes,fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates,radioactive groups, small organic molecules, polynucleotide sequencesand solid surfaces.